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Nuclear Pre-mRNA Splicing in PlantsA.S.N. Reddy aa Department of Biology and Program in Cell and Molecular Biology, Colorado StateUniversity, Fort Collins, Colorado 80523 Phone: 970-491-5773; Fax. 970-491-0649. E-mail:reddy@lamar.colostate.eduPublished online: 24 Jun 2010.

To cite this article: A.S.N. Reddy (2001) Nuclear Pre-mRNA Splicing in Plants, Critical Reviews in Plant Sciences, 20:6,523-571

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Critical Reviews in Plant Sciences, 20(6):523–571 (2001)

0735-2689/01/$.50© 2001 by CRC Press LLC

Nuclear Pre-mRNA Splicing in Plants

A.S.N. ReddyDepartment of Biology and Program in Cell and Molecular Biology, Colorado State University, Fort Collins,Colorado 80523 Phone: 970-491-5773; Fax. 970-491-0649. E-mail: reddy@lamar.colostate.edu

Referee: Dr. Gabor Lazar, Chief, Dept. of Molecular Biology, Massachsetts General Hospital, Harvard Medical School, Wellman

Eleven, Boston, MA 02114

TABLE OF CONTENTS

I. Introduction ............................................................................................................... 524

II. Structural Features of Plant Introns ...................................................................... 525

III. The Spliceosome Cycle.............................................................................................. 530A. Major (U2-type) Spliceosome .......................................................................... 530B. Minor (U12-type) Spliceosome ....................................................................... 533

IV. Plant Spliceosome...................................................................................................... 533A. Plant U snRNAs and U snRNPs ...................................................................... 535B. Serine/Arginine-Rich Proteins ......................................................................... 544

V. Intron and Exon Definition Models for pre-mRNA Recognition ........................ 551

VI. Alternative Splicing in Plants .................................................................................. 552

VII. Effect of Stresses on pre-mRNA Splicing............................................................... 559

VIII. Functions of Introns .................................................................................................. 560

IX. Conclusions ................................................................................................................ 561

X. Acknowledgments...................................................................................................... 562

XI. References ............................................................................................................................ 562

ABSTRACT: The coding regions of about 80% of plant nuclear genes contain one or more noncoding interveningsequences (introns). The transcription of these genes results in a precursor mRNA (pre-mRNA) with codingsequences (exons) and introns. The noncoding intervening sequences are then accurately removed and the codingregions are joined in the nucleus to generate functional mRNAs by a process called pre-mRNA splicing. In additionto basic/constitutive splicing, many plant pre-mRNAs, like metazoan pre-mRNAs, undergo alternative splicing,

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thereby contributing to proteomic complexity. The splicing of pre-mRNAs takes place in a large RNA-proteincomplex named the spliceosome, which is made up of several small nuclear ribonucleoprotein (snRNP) particlesand other associated proteins. Until recently, it was thought that there is only one type of spliceosome in alleukaryotes. However, it is now established that most metazoans have a second (minor) type of spliceosome thatis compositionally different from the widely studied major spliceosome and functions in splicing of some rareintrons. Based on the conservation of many components of major and minor spliceosomes in plants, it is likely thatplants contain both types of spliceosomes and the basic mechanisms involved in spliceosome formation and intronremoval are likely to be similar between plants and animals. However, a number of reports using in vivo splicingassays have shown that the cis-elements that are necessary for intron recognition and proper splicing of plantintrons differ considerably from yeast and animals. These studies suggest that the mechanisms of intron recognitionin plants are likely to differ from yeast and animals and involve novel proteins that recognize the plant-specificcis-elements. In recent years, several proteins that are implicated in plant pre-mRNA splicing have been charac-terized, including some novel ones that are not present in metazoans. The recent completion of the Arabidopsisand other eukaryotic genomes sequence should facilitate the identification of plant orthologs of various animalspliceosomal proteins in the near future. However, identification and functional analysis of the splicing proteinsthat are specific to plants will demand novel approaches.

KEY WORDS: gene expression, alternative splicing, serine/arginine-rich proteins, RNA processing, spliceosome,Arabidopsis, intron, exon, RNA helicase, snRNP.

I. INTRODUCTION

Since the first discovery of noncoding se-quences in 1977, it has been found that most eu-karyotic genes contain noncoding intervening se-quences (introns) (Berget et al., 1977; Chow et al.,1977; Goodall et al., 1991; Sharp, 1994). Intronshave been found in nuclear, chloroplast and mito-chondrial genes. It is estimated that about 80% ofall nuclear genes in plants contain introns (Goodallet al., 1991). In Arabidopsis 79% of nuclear genesand 18% and 12% of plastid and mitochondrialgenes, respectively, contain introns (Initiative,2000), and it is expected that a similar percentageof genes contain introns in other plant species. Thepre-mRNAs must be efficiently and accuratelyspliced in the nucleus to produce functional mRNAsthat are then transported into the cytoplasm fortranslation. The process by which the introns areremoved and exons are joined to generate func-tional mRNA is called RNA splicing (Sharp, 1994).Some pre-mRNAs with multiple introns displaycomplex patterns of alternative splicing (Smith etal., 1989; Lorkovic et al., 2000b; Smith andValcarcel, 2000). The splicing of pre-mRNAs isone of the important steps in RNA processing andplays a key role in regulating gene expression. It isbecoming increasingly clear that basic and alterna-tive splicing of pre-mRNAs play an important rolein development and differentiation of multicellularorganisms and in producing structurally and func-tionally different proteins from a single gene (Smith

et al., 1989; Sharp, 1994; Smith and Valcarcel,2000). Aberrant splicing has dramatic biologicalconsequences. For example, about 15% of humangenetic diseases are caused due to mutations thateither generate a new splice site or destroy thefunctional splice sites (Cooper and Mattox, 1997).In about 25 Arabidopsis mutants the mutation iseither in the 5' or 3' splice site leading to abnormalsplicing and mutant phenotype (http://www.arabidopsis.org/splice_site_mut.html). De-spite its important role in gene regulation, themechanisms that regulate basic and alternativesplicing in plants are poorly understood. Due to thelack of a plant-derived in vitro splicing system theassembly of the spliceosome is not well studied inplants. Based on the conservation of RNA andprotein components of the spliceosome in plantsand animals, it is assumed that plants have a similarspliceosome cycle. A large body of information,derived from in vivo pre-mRNA splicing studies,on the cis-elements necessary for intron recogni-tion in plants has been obtained in several labora-tories. These studies indicate that plant introns haveunique cis-elements. During the last few years sev-eral spliceosomal proteins have also been charac-terized in plants. Also, the number of plant pre-mRNAs that undergo alternative splicing isincreasing at a rapid rate. Here I summarized therecent progress on nuclear pre-mRNA splicing inplants. Several excellent comprehensive reviewson plant pre-mRNA splicing have appeared duringthe last 10 years (Goodall et al., 1991; Brown et al.,

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1993; Luehrsen et al., 1994; Simpson andFilipowicz, 1996; Brown and Simpson, 1998;Schuler, 1998; Lorkovic et al., 2000b). For theaspects that are not covered in this article, thereader is directed to these reviews. Pre-mRNAsplicing has been studied extensively in non-plantsystems and enormous progress has been made inunderstanding basic and alternative splicing inanimals. It is beyond the scope of this article toreview all the non-plant literature on splicing.However, animal-splicing literature that is relevantto plants, especially the comparison of various as-pects of splicing between plant and non-plant sys-tems, is covered here. In discussing the non-plantliterature, emphasis is given to vertebrates as splic-ing in plants is somewhat similar to these organ-isms when compared with yeast and other lowereukaryotes.

The excision of introns and joining of exonsrequires the recognition of exonic and intronic se-quences and selection of 5' and 3' splice sites. Thesequence elements (cis-elements) in the introns,exons, and at the exon/intron borders contribute tothe recognition of splice sites by a large number oftrans-acting factors. The removal of noncoding in-tron sequences from pre-mRNA and the joining ofexon sequences involves two sequential trans-es-terification reactions (Moore and Sharp, 1993; Sharp,1994). The first reaction involves a nucleophilicattack at the 5' splice site phosphate by the 2' hy-droxyl group of the adenosine in the branch point.This generates a 2' to 5' bond between the firstnucleotide of the intron and the adenosine in thebranch site upstream of the 3' splice site to form alariat structure, and a free 3' hydroxyl group on the5' exon (Figure 1). The second reaction involvesanother nucleophilic attack of the 3' hydroxyl groupon the phosphodiester bond at the 3' splice site,resulting in the release of the intron in a lariat formand ligation of the two exons (Figure 1). These tworeactions take place in a large RNA-protein complexcalled the spliceosome (Sharp, 1994).

II. STRUCTURAL FEATURES OF PLANTINTRONS

Exon/intron architecture varies across theeukaryotic kingdom. Vertebrate genes typically

have large introns and small exons, whereas theopposite is true in lower eukaryotes (Sterner etal., 1996). In general, plant genes are shorter andhave fewer introns as compared with animals.The average gene size in Arabidopsis is about 5kb, whereas it is 27 kb in humans (Initiative,2000; Venter et al., 2001). In humans, many genesare over 100 kb long. The dystrophin gene, whichis about 2.4 Mb, is the largest known gene (Con-sortium, 2001). Exons span 1.4% of the total basepairs in the human genome, whereas introns span36.4% of the base pairs (includes annotated andhypothetical genes) (Venter et al., 2001). Thehighest predicted number of introns found in theArabidopsis genome is 77 (http://mips.gsf.de/proj/thal/db/tables/tables_gen_frame.html). In contrast,the titin gene in humans with its 233 introns hasthe most introns in any organism (Venter et al.,2001). The average number of introns per gene inArabidopsis is about five, whereas it is 8.8 inhumans. Figure 2 shows the number of intronsand their distribution in the Arabidopsis genome.The average size of exons and introns in plants ismuch shorter than in animals. The sizes of thepredicted introns in Arabidopsis range from 51 to6442 nucleotides, whereas the predicted range ofexons is from 1 to 7713 (http://mips.gsf.de/proj/thal/db/tables/tables_gen_frame.html). The aver-age size of exon and introns in Arabidopsis isabout 250 and 167 bases, respectively (Initiative,2000), whereas the typical length of exons inanimals is about five to six times larger (1311 bpin C. elegans; 1497 in D. melanogaster; and 1340bp in human) (Consortium, 2001). Although thetypical length of exons is conserved in fly, worm,and humans, the length of introns is quite variableamong these organisms. In C.elegans,D. melanogaster, and humans the average lengthof an intron is 267 bp, 487 bp and 3300 bp,respectively (Consortium, 2001).

The factors that contribute to the recognitionof exons and introns are poorly understood. Theelements that confer splicing specificity must becontained within the sequences present in the in-tron and flanking exons. Analysis of nuclear pre-mRNAs from various organisms revealed threeconserved sequence elements in introns (Luehrsenet al., 1994; Sharp, 1994; Brown et al., 1996;Brown and Simpson, 1998; Schuler, 1998; Burge

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et al., 1999; Lorkovic et al., 2000b). These in-clude exon/intron boundaries at the 5' (5' splicesite/donor site) and 3' (3' splice site/acceptor site),and the branch site. The consensus sequences atthe 5' and 3' splice sites and in branch point inanimals, plants, and yeast are shown in Table 1.Plant introns, like non-plant introns, have canoni-cal GU and AG dinucleotides at the 5’ and 3’ endsof the introns and are similar to vertebrate splicesites (Brown et al., 1996; Brown and Simpson,1998; Lorkovic et al., 2000b). The branch pointsequence is mapped in only a few cases in plants(Liu and Filipowicz, 1996; Lal et al., 1999). In theArabidopsis mutant (rca), the highly conserveddinucleotide (GU) at the 5' splice site of intron 3in rubisco activase is mutated to GA, resulting inan accumulation of partially processed introns.

Using this mutant, apart from mapping the branchpoint, it was demonstrated that plant introns formintron lariat-exon intermediates (Liu andFilipowicz, 1996; Lal et al., 1999). The impor-tance of the branch point in plant pre-mRNAsplicing was confirmed by mutational analysis(Simpson et al., 1996). Insertion of a branch siteconsensus sequence in a poorly spliced intronactivated a 3' splice site downstream of the branchpoint. In addition, mutations in putative branchpoint adenosines within the consensus sequencesof four different plant introns significantly re-duced splicing efficiency of the pre-mRNA de-rived from these mutants (Simpson et al., 1996).In plants the branch point is less conserved whencompared with non-plant systems and lies about30 nucleotides upstream of the 3' intron/exon

FIGURE 1. Diagram showing two catalytic steps in nuclear pre-mRNAsplicing. A pre-mRNA with a single intron as a line and two exons (E1 andE2) is shown on the top. Two transesterification reactions (step 1 and step2) occur in the spliceosome as described in the text resulting in joining of theexons and removal of the intron in a lariat form. 5′ss, 5′splice site; BP, branchpoint, and 3′SS, 3′splice site.

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FIGURE 2. The number of introns and their distribution in the Arabidopsis genome. About 20% of thegenes have no introns and the rest have introns ranging from 1 to 77 (http://mips.gsf.de/proj/thal/db/tables/tables_gen_frame.html).

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TABLE 1Conserved Sequences at the Exon/Intron Boundary and in the Branch Point of U2-andU-12 type Introns (Burge et al., 1999; Schular, 1998; Lorkovic et al., 2000)

boundary. The branch site in plant introns re-sembles that of vertebrate introns but differs fromthe highly conserved branch point of yeast introns(Lorkovic et al., 2000b). In mammalian systems,the 3' splice site is preceded by a stretch of pyri-midines (polypyrimidine tract) whereas in plantsthe corresponding region is rich in uridines(Simpson and Filipowicz, 1996; Domon et al.,1998; Schuler, 1998).

A majority (about 99.9%) of introns are GT-AG (rarely GC-AG) or U2-type introns. Recently,a new group of nuclear pre-mRNA introns with adistinct set of consensus sequences (mostly withAT-AC termini, hence called AT-AC type in-trons) have been identified (Jackson, 1991; Halland Padgett, 1994; Dietrich et al., 1997). Theseintrons are spliced in spliceosomes that are com-positionally different from the major spliceosome(discussed below) (Hall and Padgett, 1996; Tarnand Steitz, 1996). Further analysis of several AT-AC introns has revealed that their unusual 5' and3' splice sites and a novel sequence upstream ofthe 3' splice site are highly conserved in this classof introns (Hall and Padgett, 1994). Members ofthis rare class of introns have strongly conserved

5' (/ATATCCTTT or /GTATCCTTT) and 3' (/YAC or /YAG) (where / represents the splice sitejunction) splice site sequences. These introns wereinitially referred to as AT-AC type because of thedinucleotides at the termini. However, this rareclass of introns have both AT-AC as well as GT-AG termini. In addition, some introns with AT-AC termini are spliced in the major spliceosome(Dietrich et al., 1997; Wu and Krainer, 1997).Hence, these introns are more appropriately re-ferred to as U12 type (see below) rather than AT-AC type. The U12-type introns also have a con-served branch site with a consensus sequenceTCCTTAAC (branch site is underlined), which islocated between 10 and 20 nucleotides upstreamof the 3' splice site. The polypyrimidine tractfound in GT-AG type introns is lacking in the raretype of introns. Analysis of over 53,000 con-firmed human introns has shown that 98.12% usethe canonical GT and AG dinucleotides at the 5'and 3' splice site, respectively, whereas 0.76% ofthem use the GC-AG dinucleotides (Consortium,2001). About 0.1% introns have AT-AC dinucle-otides that are recognized by U12 splicing ma-chinery (Burge et al., 1998; Consortium, 2001).

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Although the 5' and 3' splice site sequences of thevast majority (99%) of introns in Arabidopsisgenes contain the canonical GT (first two nucle-otides in the intron) -AG (last two new nucle-otides in the intron) borders there are some in-trons with noncanonical splice sites (Brown et al.,1996).

Several studies have shown that the require-ments for intron recognition during pre-mRNAsplicing in plants are different from animals andyeast. Although some structural features of plantintrons are similar to animal introns, plant intronsare unique in many aspects (Goodall et al., 1991;Lorkovic et al., 2000b). There are significant dif-ferences in cis-elements involved in splicing ofplant and vertebrate introns (Goodall et al., 1991;Simpson and Filipowicz, 1996; Brown andSimpson, 1998; Schuler, 1998). As indicatedabove, plant introns lack a distinct polypyrimidinetract near the 3' splice that is present in vertebratesand also the highly conserved branch point inyeast introns is absent in plant introns. In vitrosplicing studies in HeLa cell extracts and in vivosplicing assays with plant and animal pre-mRNAsindicate that significant differences exist in themechanisms involved in the recognition of plantand animal introns (Goodall et al., 1991; Schuler,1998). Most plant intron-containing transcripts,with some exceptions, are either not processed orprocessed inaccurately in mammalian nuclearextracts (Brown et al., 1986; Hartmuth and Barta,1986; Hartmuth and Barta, 1987; van Santen andSpritz, 1987b; Weibauer et al., 1988; Goodall etal., 1991; Schuler, 1998). Furthermore, animalintrons are not excised from pre-mRNA transcriptsin vivo in plant nuclei (Barta et al., 1986; vanSanten and Spritz, 1987b; Pautot et al., 1989;Schuler, 1998). Also, non-intron sequences ofanimal or bacterial origin are sometimes crypti-cally spliced in plants (Last et al., 1991; Haseloffet al., 1997; Schuler, 1998). These studies indi-cate that at least some of the proteins involved insplicing of plant pre-mRNAs and the mechanismsinvolved in intron recognition are likely to bedifferent from animals. Splicing studies with di-cot introns in monocot cells and vice versa indi-cate that intron recognition signals may be some-what different between dicots and monocots(reviewed in Schuler, 1998). The factors that dis-

tinguish plant pre-mRNAs from non-plant pre-mRNAs are unclear.

A distinguishing feature of plant introns istheir compositional bias for UA- or U-rich se-quences as compared to the introns from animalsand yeast (Goodall et al., 1991; Luehrsen et al.,1994; Brown et al., 1996; Simpson andFilipowicz, 1996; Ko et al., 1998). Plant intronsare generally about 15% more rich in UA, pri-marily due to an increased number of U resi-dues, than exons (Goodall and Filipowicz, 1989;Goodall et al., 1991; Luehrsen et al., 1994;Simpson and Filipowicz, 1996; Brown andSimpson, 1998; Ko et al., 1998; Schuler, 1998).In Arabidopsis, the UA content in introns isabout 67%, whereas it is only about 56% inexons (Initiative, 2000). Several reports usingsynthetic and natural plant introns have clearlyshown that intronic UA- or U-richness is impor-tant for recognition of 5’ and 3’ splice sites andfor efficient splicing of introns in plant cells(Goodall and Filipowicz, 1989; Lou et al., 1993a;Lou et al., 1993b; McCullough et al., 1993;McCullough and Schuler, 1993; Simpson andBrown, 1993; Carle-Urioste et al., 1994; Luehrsenand Walbot, 1994b; Luehrsen and Walbot, 1994a;Gniadkowski et al., 1996; Egoavil et al., 1997;McCullough and Schuler, 1997; Merritt et al.,1997; Ko et al., 1998). Although a majority ofplant introns are rich in UA or U, the dicotintrons have a higher UA content (average 74%)when compared with monocot introns (averageUA content is about 59%) (Goodall andFilipowicz, 1991). Goodall and Filipowicz (1989)first demonstrated a positive correlation betweenUA richness in introns and splicing efficiency.They have shown that synthetic introns with75% UA spliced with a very high efficiency intobacco protoplasts as compared to introns con-taining high GC. Insertion of AU-rich segmentsof maize introns near the single intron of themaize Bronze-2(Bz2) gene resulted in alterna-tive splicing (Luehrsen and Walbot, 1994b).Furthermore, insertion of AU- or U-rich se-quences into coding regions created a new intronusing splice junctions at the edges of the AU-rich region (Simpson and Brown, 1993; Luehrsenand Walbot, 1994b; Luehrsen and Walbot,1994a). In a more recent analysis of a large set

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of introns and their flanking exons from maizeand Arabidopsis, it was found that the intronsare U rich with no apparent bias for A (Ko et al.,1998). An 11-mer U-rich motif was identified asa frequent element of maize introns, which isabsent in exons (Ko et al., 1998). Altering thesequence of a U-rich motif in the maize Bronze-2 intron adversely impacted splicing, suggestingthat the U-rich motifs plays a key role in intronprocessing in vivo (Ko et al., 1998). Insertions ofa U-rich sequence (UUUUUAU) in a syntheticintron have been shown to activate splicing andmultiple U-rich segments had an additive stimu-latory effect on splicing. However, the insertionof an A-rich (AUAAAAA) sequence in a GC-rich synthetic intron did not activate splicing(Gniadkowski et al., 1996). In addition to splic-ing efficiency, the UA-rich sequences in plantintrons are required for 5' and 3' splice siterecognition (McCullough et al., 1993).Latijnhouwers et al., (1999) have tested splicingefficiency of an intron in a Bronze-2 gene wherethe composition of exon and intron is modifiedby site-directed mutagenesis. Increasing the Ucontent in the exon, whether close to or distantfrom the 5' splice site, did not modify splicingefficiency, whereas decreasing exon G+C con-tent impaired splicing. In contrast, decreasingthe content of U or increasing of GC in intronsadversely impacted splicing, suggesting that GCcontent in exons and U content in introns con-tribute to intron recognition in the Bornze 2 pre-mRNA. Inclusion of this compositional contrastfactor into statistical methods for predicting splicesites has greatly improved the accuracy of splicesite prediction in plants (Brendel et al., 1998). Adetailed discussion on various reports on theimportance of UA or U richness in plant intronsfor efficient splicing as well as for 5' and 3'splice site recognition has appeared recently(Schuler, 1998). Although several reports havedemonstrated that UA- or U-rich sequences inintrons are essential for efficient and accuratesplicing of introns in plants, it is not known howthis property influences splicing. It is likely thatplants may have protein factors that recognizethe U-rich region and participate in intron recog-nition. A few proteins that specifically interactwith U-rich intron sequences have appeared re-

cently (Gniadkowski et al., 1996; Lambermon etal., 2000), and the binding of these proteins canbe competed by poly (U) sequence but not byother primers (discussed in section III).

Although most plant introns are AU rich or Urich, about 20% of the introns in maize with over50% GC are efficiently spliced (Carle-Urioste etal., 1997). By changing exon, intron, and splicesite sequences of maize GC-rich introns, it wasfound that exon sequences also play an importantrole in intron recognition (Carle-Urioste et al.,1997). In these cases, the splicing efficiency ap-pears to be dependent on the relative difference inGC and U content between exon and intron se-quences near the splice sites, rather than the abso-lute base content of the intron or exons (Carle-Urioste et al., 1997). Other studies have also shownthat exons play an important role in intron recog-nition and/or processing (Carle-Urioste et al., 1994;Latijnhouwers et al., 1999).

III. THE SPLICEOSOME CYCLE

Two distinct types of spliceosomes have beenidentified in animal cells. The major class (U2-type spliceosome) is ubiquitous in all eukaryotes,whereas the newly discovered minor class (U12-type spliceosome) may not be universal in eu-karyotes.

A. Major (U2-type) Spliceosome

The splicing of major (GT-AG) introns hasbeen studied extensively in yeast and animal sys-tems using in vitro splicing extracts. The assem-bly of the spliceosome involves a series ofRNA-RNA, RNA-protein, and protein-protein in-teractions. Five small nuclear (sn) RNAs (U1,U2, U4, U5, and U6) (Table 2) and over 100proteins are involved in recognizing intron-exonjunctions, excision of introns, and joining of ex-ons (Burge et al., 1999). The secondary structureand short stretches of nucleotide sequences of UsnRNAs are highly conserved in all eukaryotes(Yu et al., 1999). U snRNAs complex with pro-teins to form U snRNPs. Specific regions of UsnRNAs within the snRNPs are free from protein

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and base pair with other snRNAs or the conservedsequences in the pre-mRNA. The spliceosome isformed by the ordered interaction of four smallnuclear ribonucleoproteins (snRNPs) and a num-ber of non-snRNP proteins with the pre-mRNA(Guthrie, 1991; Staley and Guthrie, 1998; Burgeet al., 1999) (Figure 3). The snRNPs recognizesplice sites and branch point sequences and aid insplicing (Guthrie, 1991; Sharp, 1994). First, theU1 snRNP recognizes the 5' splice site in an ATP-independent manner to form a complex that com-mits the pre-mRNA to spliceosome assembly.This complex is called the early (E) complex inmammalian cells or commitment complex (CC)in yeast (Figure 3). The highly conserved 10 nucle-otides at the 5' end of U1 snRNA base pair withthe 5' splice site. The association of U1 snRNPwith the 5' sequence is critical for splicing with afew exceptions (Crispino et al., 1994; Tarn andSteitz, 1994; Crispino et al., 1996; Burge et al.,1999). Then the U2 snRNP recognizes the branchsite of the pre-mRNA to form a pre-splicing com-plex (called complex A in mammals/ B in yeast).The primary function of U1 snRNP is to promoteassociation of U2 snRNP with the branch point(Seraphin and Rosbash, 1990; Burge et al., 1999).This is followed by ATP-dependent associationof tri-snRNP (U4/U5/U6) to form a complex B1(A2-1 in yeast), the assembled spliceosome. Theprogression of complex B1 through B2 involvesdissociation of U1 snRNP and U4 snRNP. Thefirst transesterification step of splicing takes placein the B2 complex and generates the lariat inter-mediate. Further progression of B2 through C1

and C2 involves ATP-dependent molecular rear-rangements. The rearrangement of RNA-RNAinteractions is facilitated by ATP-hydrolyzing non-snRNP splicing factors that have helicase-likeactivities (Staley and Guthrie, 1998). At the timeof the first catalysis only three snRNAs (U2, U5,and U6) are associated with the spliceosomes. Ofthese, U5 pairing with the exon is found to be notessential. Hence, it is likely that the catalytic sitesfor the first and second step are created by U6snRNA, U2 snRNA (or both) and/or associatedproteins (Burge et al., 1999). The release of splicedexons, lariat intron, and snRNAs (U2, U5, andU6) is facilitated by protein factors. A U5 snRNP-specific protein Prp8 (also called U5-220, p220,or hPrp8) is found to be associated with sequencesat the 5' splice site, branch point, and 3' splice site.Because of this association and the highly con-served nature of this protein, it is implicated incatalysis and/or alignment of splice sites. Thecatalytic core of the spliceosome has yet to bedefined. It is not clear which component (RNA orprotein or both) catalyzes the transesterificationreactions (Yu et al., 1999; Collins and Guthrie,2000). Although there is much evidence in sup-port of the hypothesis that catalytic steps of pre-mRNA splicing are mediated by spliceosomalRNAs, the role of protein(s) in catalysis has notbeen ruled out (Burge et al., 1999; Yu et al., 1999;Collins and Guthrie, 2000). The hydrolysis ofATP by a group of RNA-dependent ATPases(DEAD/H proteins) is required for spliceosomeassembly (Xu et al., 1996; Staley and Guthrie,1998; Schwer and Meszaros, 2000; Schwer, 2001).

TABLE 2Small Nuclear Ribonucleoproteins Involved in Nuclear pre-mRNA Splicing

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FIGURE 3. The U2-type spliceosome cycle. Sequential association of various snRNPs with a pre-mRNA results inthe assembly of a spliceosome. The letter next to each step refers to complexes that have been defined biochemi-cally and/or genetically in mammalian or yeast (in parenthesis). The non-snRNP proteins are not shown. Most ofthe components of U snRNP are highly conserved between metazoan and plant systems. 5' and 3' splice sites (5'SS and 3' SS) and branch point (BP) consensus sequences of plant U2-type introns are shown in the intron. Thefirst and second transesterification reactions are shown with an arrow in complex B2 and complex C1, respectively.For other details see the text. (Modified from Burge et al., 1999.)

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The spliceosomal snRNPs and non-snRNP pro-teins orchestrate pre-mRNA splicing. Furtherdetails on each step, rearrangements in thespliceosome and various protein factors neces-sary for spliceosome formation are reviewed inseveral recent reviews (Staley and Guthrie, 1998;Burge et al., 1999; Collins and Guthrie, 2000;Graveley, 2000; Smith and Valcarcel, 2000)

B. Minor (U12-Type Spliceosome)

The recent identification of AT-AC introns(discussed above) has led to the identification ofa new type of spliceosome. By searching the smallnuclear RNA (snRNA) sequences for regionscomplementary to the conserved elements of thisrare AT-AC class of introns, it was found that twolow abundant U11 and U12 snRNAs have a sig-nificant sequence complementarity with the 5'splice site sequence and the 3' upstream element,respectively. More recently it has been shownthat AT-AC introns are spliced in a novel type ofspliceosome that is compositionally distinct fromthe major U2-type spliceosome (Hall and Padgett,1996; Tarn and Steitz, 1996). In addition to U11and U12 snRNAs, two other novel snRNAs(U4atac and U6atac) are present in the novel (U12-type) spliceosome. The U11, U12, U4atac, andU6atac are functionally analogous to U1, U2, U4,and U6 in the major (U2-type) spliceosome. Al-though the sequences of the U11, U12, U4atac,and U6atac are different from the U2-type coun-terparts, they form similar secondary structureand in some cases the conserved core sequencesare present in both types. U5 snRNA is present inboth types of spliceosomes. Several lines of evi-dence suggests that both U2 and U12 typespliceosomes share common protein componentsand have a similar spliceosome cycle (Burge etal., 1999; Wu and Krainer, 1999; Yu et al., 1999).The rare introns are referred to as U12 type be-cause the first stable spliceosome complex isformed by the association of U12 snRNA with thebranch point (Figure 4). It is estimated that about0.1% of total introns are likely to be that of U12type. U12-type introns are present in insects, hu-man, mouse, frog, and plants, suggesting that theorigin of this minor class of introns predates the

divergence of plants and animals (Wu et al., 1996;Burge et al., 1998; Shukla and Padgett, 1999; Wuand Krainer, 1999). However, based on the analy-sis of the completed genomes U12-type intronsdo not appear to be present in S. cerevisiae and C.elegans, indicating that U12-type spliceosomemachinery may have been lost in some lineages(Burge et al., 1998; Burge et al., 1999).

Homologues of U6tatc and U12 have beenidentified in Arabidopsis, which show overallidentities of about 65% and 55% with their hu-man counterparts, respectively (Shukla andPadgett, 1999). However, the stretches of se-quences that are known to be important for theirfunction are completely conserved between plantand human U6tatc and U12 snRNAs (Shukla andPadgett, 1999). It was also shown that U6atacintramolecular stem-loop is the functional analogof the human sequence (Shukla and Padgett, 1999).Several U12-type introns were identified inArabidopsis and the splice site sequences of theseintrons are identical to the animal sequences(Burge et al., 1998; Shukla and Padgett, 1999).Detailed analysis of the recently completedArabidopsis genome should help identify the num-ber of various types of U snRNAs, including UsnRNAs of the minor spliceosome.

IV. PLANT SPLICEOSOME

The lack of an in vitro splicing system de-rived from plant cells has hampered the studies onthe assembly and biochemical characterization ofthe plant spliceosome. However, because of theconservation of many components (sequence andstructures of U snRNAs, U snRNPs, and othersplicing proteins, see below) involved in meta-zoan spliceosome assembly, it is assumed thatplants have both U2- and U12-type spliceosomesand are presumed to follow a similar assemblypathway (Solymosy and Pollak, 1993; Luehrsenet al., 1994; Schuler, 1998; Simpson et al., 1998;Shukla and Padgett, 1999; Lorkovic et al., 2000b).Because the intron recognition signals in plantsappear to be distinct from yeast and vertebrates, itis likely that the mechanisms of splice site defini-tion may be different in plants and animals. Thediscovery of novel members of serine/arginine-

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FIG

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rich proteins in plants supports this notion (seeSection IV. C).

A. Plant U-snRNAs and U-snRNPs

Small ribonucleoproteins (RNPs) are com-plexes of one or more proteins with a short RNAmolecule of about 60 to 300 nucleotides. RNPsare present in all compartments of eukaryotic cells.There are about 200 RNPs in the nucleus that fallinto two types. The ones that reside in the nucleo-plasm are called small nuclear RNPs (SnRNPs),and they function primarily in splicing (excepttelomerase snRNP, which is essential forpostreplicative addition of telomeres and genomemaintenance). The second type of RNPs reside inthe nucleolus, termed small nucleolar RNPs(SnoRNPs), and function in modification or cleav-age of pre-ribosomal RNA (Yu et al., 1999).

SnRNPs that participate in nuclear pre-mRNAsplicing contain uridine-rich RNA and the nameof the U SnRNP is derived from the type of RNAit contains. At least nine U snRNPs (U1, U2, U4,U5, U6, U11, U12, U4atac, U6atac) function innuclear pre-mRNA splicing (Table 1). The snRNPshave been isolated and extensively characterizedin animal systems. The snRNAs of U snRNPs(except U6 and U6atac) are synthesized by RNApolymerase II and are transported to the cyto-plasm. About 40 proteins associated with thesesnRNPs have been identified. Eight proteins (B,B′, D1, D2, D3, E, F, and G), called common orcore proteins, are present in all, except U6 andU6atac, spliceosomal snRNPs, and form the struc-tural core of the snRNPs. In addition to commoncore proteins, each snRNP has its own proteinsthat are unique to it (Lührmann, 1988; Andersonet al. 1991). For example, human U2 snRNP has11 specific proteins and yeast U1 snRNP has asmany as 10 specific proteins (Gottschalk et al.,1998). All snRNAs of the spliceosome except U6have unique trimethylguanosine at their 5' end.Interestingly, the core proteins are the targets ofautoantibodies present in humans and other mam-mals with autoimmune diseases. All Sm proteinsin humans have two (32 and 14 amino acids long)conserved motifs, Sm 1 and 2, which are presentin Sm proteins from diverse organisms. Sm mo-

tifs are involved in Sm protein-protein interac-tions (Hermann et al., 1995). In a comprehensiveanalysis of protein-protein interactions in yeast itwas found that each Sm protein interacted with afew other Sm proteins and a large number of otherproteins (Figure 5) (Uetz et al., 2000). For ex-ample, the yeast Lsm2, a homolog of D1, inter-acted with Lsm1 (homolog of B), smd2 (homologof D2), Lsm5 (homolog of E), and Lsm6 (ho-molog of F), Lsm 7 (homolog of G) and six otherproteins (Figure 5). This study illustrates the com-plex interactions among snRNP proteins and be-tween snRNP and other proteins (Uetz et al., 2000).

Several studies have shown that U SnRNAsand the proteins associated with them are highlyconserved among phylogenetically diverse organ-isms (Tollervey and Mattaj, 1987; van Santen andSpritz, 1987a; Hamm et al., 1988; Vankan et al.,1988; Vankan and Filipowicz, 1988; Palfi et al.,1989; Hanley and Schuler, 1991b; Hanley andSchuler, 1991a; Musci et al., 1992a; Vaux et al.,1992; Solymosy and Pollak, 1993; Simpson et al.,1995; Golovkin and Reddy, 1996; Shukla andPadgett, 1999). U1, U2, U4, U5, and U6 Sn RNPshave been purified from bean nuclear extract us-ing anti-m3G immunoaffinity chromatography(Palfi et al., 1989). The characterization of pro-teins associated with these snRNPs with anti-Smsera and other monoclonal antibodies indicatesthat plants contain the homologs of Sm proteinand other snRNP proteins (Palfi et al., 1989).Several plant hypothetical proteins also containthe conserved Sm motifs (Hermann et al., 1995),suggesting that plants have the homologs of snRNP core proteins. All the spliceosomal UsnRNAs of the major spliceosome (U1, U2, U4,U5, and U6) and some of the minor spliceosomehave been identified in Arabidopsis (Vankan etal., 1988; Vankan and Filipowicz, 1988; Hanleyand Schuler, 1991a; Hofmann et al., 1992;Solymosy and Pollak, 1993; Shukla and Padgett,1999). Plant U snRNAs are coded by large genefamilies. In Arabidopsis over 60 U snRNAs arepresent and the number of individual U snRNAsranges from 7 to 18 (http://mips.gsf.de/proj/thal/db/tables/tables_gen_frame.html). Based onSouthern analysis, it was estimated that there areabout eight or nine U5 genes in Arabidopsis(Vankan et al., 1988) and analysis of the recently

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FIG

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completed Arabidopsis genome sequence indi-cates that there are 10 U5 snRNA genes(ht tp : / /mips .gs f .de/pro j / tha l /db/ tab les /tables_gen_frame.html). Analyses of upstreamnon-coding regions of plant U snRNA genes haveshown two highly conserved sequence elements,one around the -70/-80 region and the other aroundthe –30 region (Vankan et al., 1988). The U snRNAgenes in Arabidopsis were found across all chro-mosomes either as singletons or in small groups(Initiative, 2000). Differential expression anddevelopmental regulation of all U snRNA genes,except the U6 gene, has been reported in plants(Hanley and Schuler, 1991b). Plant snRNAs showmuch greater variation in their sequences whencompared with vertebrate snRNAs (Hanley andSchuler, 1991b; Hanley and Schuler, 1991a; Musciet al., 1992a; Solymosy and Pollak, 1993). How-ever, in most of these cases variations are not inthe regions that are functionally significant. Acomparison of the plant snRNA variants withother organisms indicate that the primary sequenceof the regions that are functionally important areconserved between plants and animals (Hanleyand Schuler, 1991a; Solymosy and Pollak, 1993).Structural variations in plant U1 snRNAs occur inregions required for U1-specific proteins binding.Differences in sequence in the regions implicatedin the binding of small ribonucleoproteins(snRNPs) to snRNAs have been observed in somecases. U1 snRNAs have been isolated from bean(van Santen and Spritz, 1987a), pea (Hanley andSchuler, 1991a), tomato (Abel et al., 1989), wheat(Musci et al., 1992b), and potato (Vaux et al.,1992). As in animals, U1 snRNAs are coded bymultiple genes that are clustered in plants (Hanleyand Schuler, 1991a; Musci et al., 1992b; Vaux etal., 1992). The U1 snRNA is the most abundant ofthe snRNAs present in eukaryotic cells and playsan important role in pre-mRNA splicing (Smith etal., 1989; Guthrie, 1991). Some plant U1 snRNAgenes are differentially expressed during devel-opment (Egeland et al., 1989; Hanley and Schuler,1991b).

In metazoans, U1 snRNP contains one U1snRNA molecule and at least 11 proteins, includ-ing three U1snRNP-specific proteins (U1-70K,U1-A, and U1-C). All three U1 snRNP-specificproteins bind Loop 1 of U1 snRNA (Hamm et al.,

1988). Two U1 snRNP-specific proteins (U1-Aand U1-70K) of metazoans have been character-ized from plants (Simpson et al., 1995; Golovkinand Reddy, 1996), and a third one has beenfound in the Arabidopsis genome database(www.mips.biochem.mpg.de/cgi-bin/proj/thal/search_funcal?all+). Genetic and biochemicalanalyses have shown that the animal U1 snRNPbinds to the 5’ splice site of pre-mRNA early inthe formation of the spliceosome (Mount et al.,1983; Rosbash and Séraphin, 1991). Recognitionof the 5’ splice site by U1 snRNP involves basepairing between complementary sequences of U1snRNA and the 5’ splice site. U1 snRNP-specificproteins are required for efficient formation of acomplex between U1 snRNA and the 5’ splicesite junction (Mount et al., 1983; Heinrichs et al.,1990). There is also evidence that the U1 snRNPinteracts with the U2 snRNP during splicing (Blacket al., 1985). More recent studies also implicatethe U1 snRNP in alternative splicing (Krainer etal., 1990; Ge and Manley, 1991; Kuo et al., 1991).As in the case of U1 snRNA, this binding alsoinvolves base pairing between highly conservedsequences in U2 and pre-mRNA. U2 auxiliaryfactor (U2AF) binds to the polypyrimidine tractbetween the branch point and the 3' splice site andis essential for the selection of the 3' splice site(Figure 6). U2AF recruits the U2 snRNP to thebranch point and is thought to be involved inbridging interactions between 5' and 3' splice sites.U2AF functions as a heterodimer with one large(U2AF65) and one small (U2AF35 subunit). TheU2AF65 and U2AF35 subunits recognize thepolypyrimidine tract and 3' AG, respectively(Moore, 2000). U2AF65 interacts with the branch-point-binding protein (BBP, also called SF1, splic-ing factor 1), which recognizes the branch site(Berglund et al., 1997). It also recruits UAP56(U2AF associated protein 56), a member of theDExD/H box protein family of RNA-dependentATPases (Fleckner et al., 1997; Zhang and Green,2001).

The Arabidopsis U1 snRNA is about 70-75%identical to other plant U1 snRNAs. TheArabidopsis U1 snRNA, like other plant and ani-mal U1 snRNAs, folds into a secondary structurethat consists of four stem-loops (I to IV). The sizeof plant U1 snRNAs varies between 157 and 165

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FIGURE 6. Spliceosome assembly in plants based on various plant spliceosomal components that have beencharacterized. A pre-mRNA with a single intron with consensus sequences of plant introns is shown. The U snRNPsand other non-snRNP proteins shown in the diagram have been identified in plants and are highly conservedbetween plants and metazoans. The placement of various snRNPs and other proteins on the pre-mRNA is basedon the information from vertebrate systems. Filled circles on the intron represent proteins that bind U-rich sequencesin the introns. SF1/BBP, splicing factor 1/branch point binding protein; m7Gppp, cap at the 5’ end; U2AF65, U1auxiliary factor 65-kDa subunit; U2AF35, U1 auxiliary factor 35 kDa subunit; SR proteins are not shown in this figure.

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nucleotides and several variants are found in agiven species (Solymosy and Pollak, 1993). Inanimals, the stem-loop I of U1 snRNA is the siteof interaction with the U1-70K protein wherefour nucleotides (27U, 29A, 30U, and 31C) arerequired for this interaction (Lührmann et al.,1990; Yuo and Weiner, 1990). These nucleotidesare present in the Arabidopsis U1 snRNA(Golovkin and Reddy, 1996). However, in someplant U1 snRNAs the nucleotide at position 27 isa C residue. It is also known that many U1 snRNAsof plants have either shorter loop sequences orlack one of the nucleotides required for binding(van Santen and Spritz, 1987a; Abel et al., 1989;Hanley and Schuler, 1991a; Vaux et al., 1992).Hence, it is possible that variant U1 snRNAs bindto the U1-70K protein with different affinities.

U1 and U2 snRNPs play important roles dur-ing the early stages of spliceosome formation.The first U snRNP protein to be characterizedfrom plants was U2B’’ from potato, which inter-acts with the conserved loop IV of U2 snRNA(Simpson et al., 1991). The plant U2B’’ is highlyconserved between plants and animals and showsextensive similarity with the human U2B’’ pro-tein, suggesting the conservation of U2B’’ func-tion in plants and metazoans. The binding site forU2B’’ on U2 snRNA is conserved between dicotsand monocots. U2B’’, as expected of an snRNPprotein, is present in the nucleus and the firstRNP-80 motif is sufficient for its localization tothe coiled body (Boudonck et al., 1999). InArabidopsis there are two Prp8-like proteins thatare highly conserved between plants and animals.Two Arabidopsis proteins are 92% similar to eachother and share 79% and 69% similarity withhuman and yeast (S. pombe) Prp8 (Figure 7).

The U1-A protein from Arabidopsis and po-tato also has a considerable sequence identitywith human U1-A (Simpson et al., 1995). Likeother RNA binding proteins, U1A, U2B” and U-70K have one or more RNA binding domains(one in U1-70K and two in U1A and U2B”), eachwith RNP (ribonucleoprotein) consensus se-quences (Burd and Dreyfuss, 1994; Simpson etal., 1995). The plant U1-A has been shown tobind specifically with plant or human U1 snRNA.In in vitro RNA binding assays potato U2B” in-teracted with human U2A’ and this complex in-

teracted with both potato or human U2 snRNA(Simpson et al., 1995).

It was shown that U snRNA and U2B arepresent in coiled bodies, nuclear organelles thatcontain splicing snRNPs. The number of coiledbodies is developmentally regulated in theArabidopsis root epidermis (Beven et al., 1995;Boudonck et al., 1998). The dividing cells have alarger number of coiled bodies when comparedwith quiescent cells, initial cells, and cells in theelongation and differentiation zone. The dynam-ics of coiled bodies was studied in living plantcells using stably expressed green fluorescentprotein fused to U2B. These studies have shownthat the coiled bodies are highly mobile, theirmovements were observed in the nucleolus, in thenucleoplasm, and from the periphery of the nucleusinto the nucleolus (Boudonck et al., 1999).

The U1-70K protein has been characterizedfrom yeast, vertebrates, and invertebrates(Theissen et al., 1986; Spritz et al., 1987; Etzerodtet al., 1988; Hornig et al., 1989; Query et al.,1989; Mancebo et al., 1990). The human U1-70Kprotein has several domains that are functionallyimportant (Figure 8). These include (1) one RNArecognition motif (RRM) containing consensusRNP2 and RNP1 sequences and a nuclear local-ization signal, (2) a glycine rich region next toRRM, (3) two arginine/serine (RS) -rich regions(residues 231–310 and 350–383), and (4) a gly-cine/proline-rich region in front and downstreamof the second RS-rich region (Woppmann et al.,1993; Romac et al., 1994). The RS-rich regionscontain several dipeptide repeats of RD/RE andRS. The C-terminal half of U1-70K contains highlycharged arginine/serine-rich regions that contrib-ute to the abnormal migration of this 52K proteinon SDS-polyacrylamide gels (Query et al., 1989).Among animals, the U1-70K protein is highlyconserved, with 88% amino acid identity betweenthe Xenopus and human proteins. However, theyeast homolog of U1-70K has only 30% overallamino acid identity with metazoans (Smith andBarrell, 1991).

The Arabidopsis U1-70K protein is encodedby a single gene and produces two transcripts(short and long) by alternative splicing of the U1-70K pre-mRNA (Goiouking and Reddy, 1996).The deduced amino acid sequence from the short

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transcript has strong homology to the animal U1-70K protein and contains an RNA recognitionmotif, a glycine hinge, and an arginine-rich re-gion characteristic of the animal U1-70K protein(Figure 9). The U1-70K gene contains nine ex-ons. The two cDNAs are produced by alternativesplicing involving inclusion or exclusion of a 910-bp intron. Both transcripts are expressed in alltissues tested and the level of the transcripts var-ied in different organs. The human U1-70K genecontains 11 exons and is representative of animalU1-70K genes because the general structure ofthe gene is highly conserved during vertebrateevolution. Conserved features include the numberand position of introns as well as the nucleotide

sequence of an alternative included/excluded exoncontaining an in-frame translation terminationcodon. Because of the conservation of the loca-tion and the sequence of the alternatively splicedincluded/excluded exon among vertebrates, it hasbeen suggested that alternative splicing of theincluded/excluded exon may regulate the produc-tion of functional U1-70K protein. The positionsof four introns (4, 5, 6, and 7) including thealternatively spliced introns are conserved be-tween Arabidopsis and human U1-70K. How-ever, intron 6 is alternatively spliced inArabidopsis, and intron 7 is alternatively splicedin human. The long transcript in Arabidopsis hasan in-frame translational termination codon within

FIGURE 7. A U5 snRNP-specific protein (Prp8/U5-220/p220) is highly conserved betweenplant and non-plant systems. (A) Phylogenetic tree showing the relationship between Prp8from different organisms. (B) Percent similarity between yeast, animal and plant Prp8 pro-teins.. Full-length proteins from various organisms were aligned using the CLUSTAL methodin the Megalign program and the aligned sequences were used to build a phylogenetic tree.

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FIGURE 8. Structural features of human SR (A) and SR-related proteins (B). RRM, RNArecognition motif; RRMH, RRM homology; Z, zink knuckle/zink finger; RS, arginine/serine-rich region; DEXD/H box, a domain present in RNA helicases. (Redrawn fromGraveley, 2000.)

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FIGURE 9. Schematic diagram showing the structural features of various Arabidopsis spliceosomal proteins andprotein kinases that phosphorylate SR proteins. RRM, RNA recognition motif; GH, glycine hinge, Z, zink knuckle;RS, arginine/serine-rich region; PSK, a domain rich in proline, serine and lysine; Q, glutamine-rich region; Q/P,glutamine and proline-rich region, RD, dipeptide repeats containing arginine and aspartic acid; GR, glycine-richregion; PKD, protein kinase domain, L, LAMMER motif in Clk/Sty protein kinases. Glycine hinges are not shown insome proteins.

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the 910-bp included intron, resulting in a trun-cated protein containing only 204 amino acidswith part of the RRM containing RNP2 only. It isof interest to note that a smaller (60 or 72 nucle-otides) alternatively spliced included/excludedexon is found in the human U1-70K gene at adifferent location within the RRM and produces atruncated protein due to the presence of an in-frame translation termination codon. However,unlike the Arabidopsis U1-70K gene, the trun-cated protein from the human U1-70K gene hasboth RNP2 and RNP1 consensus sequences. It ispossible that the long transcripts with the includedintron may be performing one or more functions.The truncated protein may regulate the amount offunctional protein in the cell and/or associate withU1 snRNP in vivo but with a different functionthan the full-length protein. Recent reports fromstudies with yeast and humans suggest this possi-bility (Nelissen et al., 1994; Hilleren et al., 1995).The N terminal region (amino acids 1 to 97) ofhuman U1-70K that lacks the RRM associatesefficiently with core U1 snRNPs (Nelissen et al.,1994), although this protein is not known to inter-act directly with U1 snRNA under in vitro condi-tions. In yeast, recently it has been shown that theN terminal region of about 92 amino acids thatdoes not contain an RNA recognition motif, butcan associate with U1 snRNP in vivo, is neces-sary, and sufficient for U1-70K function (Hillerenet al., 1995). The truncated Arabidopsis proteinencoded by the large transcript shows significantsequence similarity (40% identity and 60% simi-larity) with the 97 amino acid N-terminal regionof human and yeast U1-70K, which is shown toassociate with U1 snRNP (Figure 10).

Although the C terminus of the ArabidopsisU1-70K protein has an arginine-rich region, it hasless sequence similarity with metazoan U1-70Kprotein. The presence of two distinct arginine/serine-rich regions in the C-terminal region is notobvious in the plant U1-70K protein. In addition,the arginine-rich region in Arabidopsis containsmostly RD/RE dipeptides (21 repeats). This mayreflect some variation in U1-70K function in plantsbecause this region in the animal protein containsmore RD/RE dipeptides (25 repeats) and RS dipep-tides (11 repeats) that interact with similar RS-regions in splicing factors. The serine residues in

the RS region are the sites of phosphorylation.The human U1-70K protein is phosphorylated atmultiple sites in the C-terminal RS region, therebygenerating 13 different variants (Woppmann etal., 1993). It has been shown that phosphorylationof U1-70K plays an important role in pre-mRNAsplicing (Tazi et al., 1993). The fact that the argi-nine-rich region in the Arabidopsis U1-70K pro-tein does not contain many RS dipeptides sug-gests that it is either not a phosphoprotein or notas heavily phosphorylated as in human U1-70Kprotein. Nevertheless, the presence of an argin-ine-rich region in Arabidopsis also suggests thatthis region may be involved in interacting withsplicing factors involved in basic and alternativesplicing. The glycine/proline-rich region betweentwo RS domains as well as a similar region at theC terminus of the second RS domain are notpresent in Arabidopsis U1-70K. Overall, theArabidopsis U1-70K protein shares certain fea-tures with the animal U1-70K protein but differsin others. These differences may reflect varia-tions in pre-mRNA splicing between plants andanimals. The protein encoded by the short cDNAis recognized by U1(RNP) monoclonal antibod-ies and binds to Arabidopsis U1 snRNA, suggest-ing that it is an ortholog of metazoan U1-70K.

Two homologs (U2AF65a and U2AF65b) ofthe large subunit of U2AF (U2AF65) have beenisolated from tobacco and both these proteins canrescue the splicing activity of HeLa cell nuclearextracts that are depleted of the endogenousU2AF65 (Domon et al., 1998). These proteins bindRNA fragments containing plant introns and showaffinity for poly(U) and, to a lesser extent, poly(C)and poly(G). The branch point or the 3' splice siteregions do not contribute significantly to intronrecognition by NpU2AF65. The homologs of to-bacco U2AF65a and U2AF65b have been identifiedin Arabidopsis. The existence of multiple isoformsof U2AF may be quite general in plants becausetwo genes expressing U2AF65 have been identi-fied in Arabidopsis, and different isoforms of theU2AF small subunit are expressed in rice. In rice,there are two genes encoding the small subunit ofU2AF35 (U2AF35a and U2AF35b), and one of thegenes undergoes alternative splicing to produceseveral isoforms of U2AF small subunit tran-scripts (Domon et al., 1998). These results sug-

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gest that plants, unlike metazoans, contain mul-tiple U2AF large and small subunits. The primarysequence and domain organization of U2AF sub-units are highly conserved between plants andanimals (Figures 8 and 9) except that the N-termi-nal region of plant U2AF subunits has a strongbias for RD over RS dipeptides. Plants also havehomologs of UAP56 and BBP/SF1(Lorkovic etal., 2000b).

A protein, UBP1, that can bind U-rich intronand 3'-UTR sequences in vitro has been charac-terized from tobacco (Lambermon et al., 2000).The UBP1 shares several features with an hnRNPprotein (Figure 9). This protein has been shown toassociate with nuclear poly(A)+ RNA in vivo andinfluence pre-mRNA splicing as well as tran-script accumulation. Overexpression of UBP1 inprotoplasts enhanced the splicing of pre-mRNAsthat are otherwise inefficiently processed and in-creased accumulation of reporter gene transcriptsthat contain suboptimal introns or no introns(Lambermon et al., 2000). Because the effects ofUBP1 on pre-mRNA splicing and accumulationare separable, it was suggested this protein maycontain two independent activities (Lambermonet al., 2000).

Recently, Lorkovic et al. (Lorkovic et al.,2000a) have isolated two structurally related RNA-binding proteins (RBPs), RBP45 and RBP47, fromtobacco (Nicotiana plumbaginifolia) that specifi-cally bind oligouridylates. The N-terminus of theseproteins is rich in glutamine and each protein hasthree RBD-type RNA-binding domains with RNP1and RNP2 consensus sequences, and show simi-larity with Nam8p, a protein associated with U1snRNP in the yeast S. cerevisiae. The first two

RBDs are adjacent to each other and the third oneis separated by a spacer (Figure 9). Both of theseproteins are localized to the nucleus. RNA bind-ing studies with truncated proteins have shownthat at least two RBDs are required for their inter-action with RNA, whereas the domains other thanRBD do not significantly contribute to binding(Lorkovic et al., 2000a). RBP45 and RBP47 arelocalized in the nucleus. Several proteins that arerelated to these proteins have been identified inArabidopsis thaliana and are found to be consti-tutively expressed in different plant organs(Lorkovic et al., 2000a). In vivo UV cross-linkingexperiments have shown that these proteins asso-ciate with the nuclear poly(A)+ RNA. UBP1,RBP45 and RBP47 have similar biochemical prop-erties and structural features. However, unlikeUBP1, overexpression of these proteins in proto-plasts has no effect on mRNA splicing and accu-mulation. Although the characteristics of theseRNA binding proteins imply a role in pre-mRNAprocessing, their precise function is yet to be de-fined. RBDs 2 and 3 of RBP45 and RBP47 areclosely related to the RBDs of yeast U1 snRNPprotein Nam8p, which plays a role in splicing ofsuboptimal introns (Gottschalk et al., 1998; Puiget al., 1999).

B. Serine/Arginine-Rich Proteins

Serine/arginine-rich (SR) proteins, a largefamily of proteins, are one of the best-character-ized non-snRNP proteins in the spliceosome. Themembers of this family have multiple roles inconstitutive and alternative splicing of pre-mRNAs

FIGURE 10. Alignment of the N-terminal region of yeast and plant U1-70K protein. Amino acids are groupedaccording to Hilleren et al. (1995). Identical (in bold) and similar amino acids between plant and yeast U1-70K areshown in reverse lettering.

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in metazoans (reviewed in Graveley, 2000). SRproteins are involved in a network of protein-protein interactions primarily through their RSdomain. These proteins, with a molecular massranging from 20 to 75 kDa, contain one or twoRNA binding domains (RBDs) and an arginine/serine-rich (RS) domain with multiple RS dipep-tide repeats at the C-terminus (Fu, 1995; Manleyand Tacke, 1996). SR proteins from animals canindividually complement splicing deficient S100extracts that contain all factors necessary for splic-ing except the SR proteins (Manley and Tacke,1996; Graveley, 2000). All SR proteins are recog-nized by a monoclonal antibody (mAb104). Inhumans there are at least 10 SR proteins andseveral SR protein-related polypeptides (Graveley,2000) (Figure 8). However, SR proteins are notfound in all eukaryotes. In budding yeast (S.cerevisiae) there are no SR proteins, whereas fis-sion yeast (S. pombe) has only two SR proteins(Graveley, 2000). SR proteins play central rolesin both constitutive and alternative splicing asessential splicing factors and as specific splicingregulators at multiple stages in spliceosome as-sembly (Manley and Tacke, 1996; Valcárcel andGreen, 1996; Caceres et al., 1997; Graveley andManiatis, 1998; Graveley et al., 1999). These pro-teins recruit other factors during spliceosome as-sembly through protein-RNA or protein-proteininteractions involving their RS domain (Graveleyand Maniatis, 1998; Graveley et al., 1999;Graveley, 2000). During E- complex formation,ASF (alternative splicing factor)/SF2 (splicingfactor 2), one of the SR proteins, recruits U1snRNP to the 5’ splice by interacting simulta-neously with the pre-mRNA and the U1-70K pro-tein (Kohtz et al., 1994). SR proteins (e.g., SC35and ASF/SF2) are also involved in bridging 5’and 3’ splice sites by interacting concurrentlywith U1-70K and U2AF35 (Wu and Maniatis, 1993;Will and Luhrmann, 1997; Stark et al., 1998).Furthermore, SR proteins facilitate incorporationof the tri-snRNP complex (U4/U6.U5 snRNP)into the spliceosome and promote base pairingbetween U2 and U6 snRNA (Roscigno and Garcia-Blanco, 1995; Tarn and Steitz, 1995). Recently,the RS domain of SR proteins has been found tomodulate RNA-RNA interactions directly(Valcárcel et al., 1996; MacMillan et al., 1997).

In addition to their role in constitutive splicing,SR proteins have been shown to play an impor-tant role in alternative splicing by influencing 5’and 3’ splice sites selection in vitro and in vivo(Lavigueur et al., 1993; Zahler et al., 1993; Cacereset al., 1994; Fu, 1995; Ramchatesingh et al., 1995;Zahler and Roth, 1995; Kanopka et al., 1996;Manley and Tacke, 1996; Caceres et al., 1997;Kanopka et al., 1998; Wang et al., 1998b). Re-cently, one of the SR proteins (SRp20) has beenshown to regulate alternative splicing of its ownpre-mRNA (Jumaa and Nielsen, 1997).

It has been demonstrated that U1-70K proteinbinds to stem-loop I at the 5’ end of the U1snRNA (Query et al., 1989). Furthermore,overexpression of the arginine/serine-rich regionof the U1-70K has been shown to inhibit splicingas well as nucleocytoplasmic transport of mRNA(Romac and Keene, 1995). These studies indicatean important role for U1-70K in splicing. In ani-mals, U1 snRNP participates in splice site selec-tion by interacting with two SR proteins (SC35and ASF/SF2), and this interaction takes placethrough a specific association of the RS domainin SR proteins and a similar region in U1-70Kprotein in the U1 snRNP (Wu and Maniatis, 1993;Kohtz et al., 1994; Manley and Tacke, 1996; Xiaoand Manley, 1997). Recently, it has been shownthat animal U1-70K interacts with Sip1 (SC35-interaction protein 1), which also contains an RSdomain but does not contain an RNA-bindingmotif (Zhang and Wu, 1998).

Several different approaches have been usedto identify plant serine/arginine-rich proteins.These include (1) the use of monoclonal antibod-ies to detect similar proteins in plant extracts,(2) salt-precipitation of plant soluble proteins toenrich SR proteins and testing their activity in SRprotein-deficient S-100 extract from HeLa cells,(3) PCR-based cloning of plant proteins using theprimers corresponding to conserved domains inanimal SR proteins, (4) Arabidopsis genome se-quence database searches with the animal SR pro-teins, and (5) screening of yeast two-hybrid li-braries with snRNP proteins. These approacheshave resulted in identification of several plantserine/arginine-rich proteins including severalnovel ones (Figure 9). So far nine SR proteinshave been reported from Arabidopsis (Lazar et

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al., 1995; Lopato et al., 1996b; Golovkin andReddy, 1998; Golovkin and Reddy, 1999; Lopatoet al., 1999a). In addition, a few SR-related pro-teins have also been characterized. Of these, somehave no real homologs in animals and some aresimilar to animals SR proteins with novel features(Lazar et al., 1995; Lopato et al., 1996b; Golovkinand Reddy, 1998; Golovkin and Reddy, 1999).Two plant SR proteins have been shown to comple-ment splicing deficient S100 extract (Lopato etal., 1996b; Lopato et al., 1999a), whereasArabidopsis ASF/SF2-like protein did not (Lazaret al., 1995).

Using a monoclonal antibody to a phos-phoepitope shared by the animal SR family ofsplicing factors Lazar et al., (Lazar et al., 1995) andLopato et al., (Lopato et al., 1996a) have shownthat plants contain a serine-arginine (SR)-rich pro-tein family. The plant SR proteins, like animalcounterparts, can be enriched by a two-step saltprecipitation (Lazar et al., 1995; Lopato et al.,1996a). However, plant SR proteins showed a com-plex pattern of intra- and interspecific variants(Lopato et al., 1996a). A mixture of plant SR pro-teins restored splicing competency of HeLa cellcytoplasmic S100 extracts that are deficient in SRproteins (Lopato et al., 1996a). Arabidopsis SR1,the first SR protein to be characterized from plants,showed significant sequence and structural homol-ogy to the human splicing factor SF2/ASF (Lazaret al., 1995). However, a novel C-terminal do-main containing a high concentration of proline,serine, and lysine residues (PSK domain) with apredicted phosphorylation site for the cyclin/p34cdc2 is not present in the animal SF2/ASF.Although SR1 does not complement S100 splic-ing extract, it promotes splice site switching ofβ−globin intron with two 5' splice sites in mam-malian nuclear extracts (Lazar et al., 1995). TheSR1 pre-mRNA undergoes alternative splicingand produces at least five (SR1A, SR1B, SR1C,and SR1D and SR1E) transcripts. One of thesetranscripts encodes the full-length protein, whereasthe other four are different variants of the essen-tial arginine-serine-rich domain (Lazar andGoodman, 2000). Another SR-like protein, namedatSRp30, with significant sequence similarity tohuman SF2/ASF was also characterized fromArabidopsis (Lopato et al., 1999b). Like, SR1/

atSRp34, the pre-mRNA of atSRp30 is alterna-tively spliced and splice variants are differentiallyexpressed in various organs and during develop-ment. Overexpression of atSRp30 results inchanges in alternative splicing of several endog-enous plant genes, including splicing of its ownpre-mRNA. Furthermore, overproduction ofatSRp30 significantly reduced the amount of en-dogenous mRNA encoding full-length SR1/atSRp34 protein and affected developmental phasetransitions (Lopato et al., 1999b). atSRp30 andSR1/atSRp34 showed complementary expressionpatterns during early seedling development androot formation with overlapping expression infloral tissues (Lopato et al., 1999b). In addition toSR1/atSRp34 and atSRp30, Lopato et al. (Lopatoet al., 1996b) identified a novel group of SRproteins (atRSp31, atRSp35 and atRSp41) fromArabidopsis (Lopato et al., 1996b). These pro-teins are similar to animal SR proteins in that theycontain an N-terminal RNA recognition motif anda C-terminal RS domain enriched in arginines.However, the region in the middle has no se-quence similarity to known protein. Bacteriallyexpressed recombinant atRSp31 restored splicingactivity in SR protein-depleted HeLa S100 ex-tracts (Lopato et al., 1996b). These genes aredifferentially expressed in a tissue-specific man-ner.

Yeast two-hybrid screening with either full-length or the C-terminal region of U1-70K hasresulted in isolation of four serine/arginine-richproteins (SRZ-22, SRZ-21, SR33, and SR45)(Golovkin and Reddy, 1998; Golovkin and Reddy,1999). These proteins share several features withSR proteins, including modular domains typicalof splicing factors in the SR family of proteins.SRZ21 and SRZ22 interact only with the full-length U1-70K, whereas SR33 and SR45 interactwith the arginine-rich region of U1-70K. This isin contrast to animal U1-70K that is known tointeract with two SR proteins (SC35 and ASF/SF2). Both of these animal SR proteins interactvia the RS domain of U1-70K (Wu and Maniatis,1993; Kohtz et al., 1994). In addition, sip1 (SC35-interaction protein 1), which also contains an RSdomain but does not contain an RNA-bindingmotif also interacts with animal U1-70K (Zhangand Wu, 1998). The plant SRZ proteins are highly

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similar to each other and contain conserved modu-lar domains unique to different groups of splicingfactors in the SR family of proteins. SRZ proteinsare similar to human 9G8 splicing factor becausethey contain a zinc knuckle, precipitate at 65%ammonium sulfate, and cross-react with the 9G8monoclonal antibody (Cavaloc et al., 1994). Theserine/arginine-rich region in the SRZ proteinsdiffered from 9G8 splicing factor in lacking theconsensus sequences (RRSRSXSX) (Cavaloc etal., 1994). Both SRZ proteins, however, contain aglycine-rich region (glycine hinge) that is presentin SF2/ASF, RSp55, SC35, and SR1 splicing fac-tors but not in 9G8 (Manley and Tacke, 1996).SRZ-22 and SRZ-21 are encoded by two distinctgenes and are expressed in all tissues tested withvaried levels of expression. These unique struc-tural features of the SRZ proteins and the fact thatthe interaction occurs with the full-length U1-70K indicate that the SRZ proteins represent anew group of serine/arginine-rich proteins. SRZproteins have several potential nuclear localiza-tion signals, including four bipartite motifs ineach SRZ protein, suggesting that these are nuclearproteins. The interaction of plant U1-70K withthe novel plant SR proteins may account for somedifferences in pre-mRNA splicing between plantsand animals. SR33 and SR45 are rich in prolineand SR45, unlike most animal SR proteins, hastwo distinct RS domains separated by an RNArecognition motif and has no sequence similarityto known SR protein. The animal U1-70K has notbeen shown to interact with any double RS do-main-containing protein. Furthermore, in vivo andin vitro protein-protein interaction experimentshave shown that SR33 protein interacts with itselfand with SR45 protein but not with two othermembers (SRZ21 and SRZ22) of the SR familythat are known to interact with the Arabidopsisfull-length U1-70K only (Figure 11).

Of the four plant SR proteins that interactwith the U1-70K, one (SR33) appears to be simi-lar to SC35 and the interaction of the other threeproteins with the U1-70K is unique to plants.Among four plant U1-70K interacting proteins,SRZ22 (also called atRSZp22) has been shown tocomplement splicing deficient HeLa cell S100extract (Lopato et al., 1999a). In animals there issingle ASF/SF2 that interacts with U1-70K. Al-

though there are at least two ASF/SF2-like pro-teins present in Arabidopsis (Lopato et al., 1999b),none of these was isolated in the yeast two-hybridscreening with either full-length or C-terminalU1-70K. Unlike its animal counterpart,Arabidopsis ASF/SF2 does not complement S100extract (Lazar et al., 1995). It would be interestingto test the interaction between plant ASF/SF2proteins with plant U1-70K. These studies dem-onstrate that plant U1-70K interacts with a differ-ent set of SR proteins, including some novel SRproteins, suggesting that early stages ofspliceosome formation or splice site selection maydiffer from animals. These data support the obser-vations that intron recognition in plants is likelyto differ from animals (Simpson and Filipowicz,1996; Schuler, 1998). Wu and Maniatis (Wu andManiatis, 1993) have shown that two SR proteins(SC35 and ASF/SF2) that interact with U1-70Kprotein can simultaneously interact with U2AF35

(U2 snRNP auxillary factor 35). Two differentforms of U2AF35 have been identified in plants(Domon et al., 1998). At present it is not knownif U1-70K interacting SR proteins associate witha similar factor in plants.

Unlike known SR proteins, SR33 and SR45proteins are rich in proline content (12% in SR33and 17% in SR45) (Golovkin and Reddy, 1999).The majority of these residues are located to-gether with the serine and arginine residues withinthe RS domains. There is a long stretch of sevenproline residues at the C-terminus of SR45(Golovkin and Reddy, 1999). A recently describedhuman splicing coactivator, SRm160, whose en-tire ORF is an arginine/serine-rich domain with-out an RBD also contains a high amount of pro-line (16%) (Blencowe et al., 1998). In addition toproline richness, SR33 and SR45 proteins containthe highest percentage of serine and arginine resi-dues (39 to 43%) among all plant SR proteins. Of10 human SR proteins, five contain one RRM,whereas the others have two such domains (Fig-ure 9). Among the SR proteins characterized inArabidopsis, six of the nine contain one RBD.

A number of recent studies indicate the im-portance of the phosphorylation status of SR pro-teins in the regulation of splicing process (Misteliet al., 1997; Misteli and Spector, 1997; Tacke etal., 1997; Xiao and Manley, 1997; Wang et al.,

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1998a; Xiao and Manley, 1998; Misteli, 1999;Yeakley et al., 1999). Several recent studies sug-gest that phosphorylation of SR proteins is re-quired for spliceosome assembly and splice siteselection (Mermoud et al., 1994; Cao et al., 1997).Furthermore, dephosphorylation of SR proteins isalso necessary for the later stages of splicing,suggesting that the phosphorylation and dephos-phorylation cycle of SR proteins plays a criticalrole in splicing (Mermoud et al., 1994; Cao et al.,1997; Murray et al., 1999). All SR proteins inanimals are phosphoproteins and can be detectedby a monoclonal antibody (mAb 104) that recog-nizes a phosphoepitope within the RS domain(Roth et al., 1990; Zahler et al., 1992; Manley andTacke, 1996). Several protein kinases that arecapable of phosphorylating serine residues in theRS domain of SR proteins have been identified(Gui et al., 1994b; Colwill et al., 1996a; Duncanet al., 1998; Kuroyanagi et al., 1998; Wang et al.,1998a). Of these, two families of protein kinases,

SRPK (SR protein-specific kinase) and Clk/Sty,which differ in their substrate specificity havebeen characterized extensively (Colwill et al.,1996a; Colwill et al., 1996b). SRPKs are presentin both the cytoplasm and the nucleus, whereasClk/Sty, a dual-specificity protein kinase that canphosphorylate serine, threonine, and tyrosine resi-dues (Colwill et al., 1996a; Colwill et al., 1996b),is present exclusively in the nucleus. The Clk/Stykinases (also called LAMMER-type kinases) con-tain a unique sequence (EHLAMMERILGDLA)in subdomain X of the kinase catalytic domain(Figure 8). The interaction of Clk/Sty with someof its target proteins involves an RS-rich region atthe N-terminus of Clk/Sty kinase. Several studiesindicate that phosphorylation of SR proteins af-fects their mode of interaction with other pro-teins. Phosphorylation of ASF/SF2 in the RSdomain enhances their interaction with U1-70Kprotein and affects their splicing activity (Xiaoand Manley, 1997; Xiao and Manley, 1998). Re-

FIGURE 11. Schematic diagram showing the interaction of Arabidopsis U1-70Kwith SR proteins. RRM. RNA recognition motif; RS, serine/arginine-rich region;AFC2, Clk/Sty-type kinase. SRZ21 and SRZ2 interact with only full-length U1-70Kwhereas SR33 and SR45 interact with the C-terminal arginine-rich region of U1-70K. The thickness of the arrows indicates interaction strength. (Modified fromGolovkin and Reddy, 1999.)

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cruitment of SR proteins to sites of transcriptionis also mediated by phosphorylation (Gui et al.,1994a; Colwill et al., 1996b; Caceres et al., 1997;Duncan et al., 1998; Misteli, 1999). SRPK andClk/Sty can influence the distribution of SR pro-teins within the nucleus and an excess of SRPKcan inhibit splicing (Gui et al., 1994a; Colwill etal., 1996b). Furthermore, nuclear import of SRproteins is also regulated by phosphorylation(Yeakley et al., 1999).

Little is known about the plant kinases thatphosphorylate plant SR proteins. Three kinasesthat are similar to Clk/Sty kinase have been iden-tified in the Arabidopsis genome (Bender and Fink,1994). One of these (AFC1) was isolated as asuppressor of mating defect in fus3 and kss1 signaltransduction mutant in S. cerevisiae. The other two(AFC2 and AFC3) were also isolated fromArabidopsis using primers corresponding to con-served regions in AFC1 kinase (Bender and Fink,1994) (Figure 9). AFC2, although closely relatedto AFC-1, does not suppress the mating defect indouble MAP kinase mutant, suggesting a differ-ence in their function. The substrates of plant Clk/Sty kinases have not been identified. One of thekinases (AFC2) undergoes autophosphorylationand heavily phosphorylated four plant SR pro-teins (SRZ21, SRZ22, SR33, and SR45) (Golovkinand Reddy, 1999). Coprecipitation studies haveconfirmed the interaction of SR proteins withAFC2 kinase and the interaction between AFC2and SR33 is modulated by the phosphorylationstatus of these proteins. The interaction betweenthe SR proteins and AFC2 is modulated by phos-phorylation. However, the plant Clk/sty kinasedoes not contain an RS-rich region in theN-terminus, suggesting that structural featuresother than an RS domain are also important in theinteraction of AFC2 with other proteins (Figures8 and 9). AFC1, which rescues yeast double MAPkinase mutant does not phosphorylate SR pro-teins, suggesting that the ability to phosphorylateSR proteins is not likely to be a feature of all Clk/Sty kinases in Arabidopsis. A member of theLAMMER family of protein kinases from to-bacco (PK12) is induced by ethylene (Sessa et al.,1996). PK12 binds and phosphorylates both plantand animal SR proteins in vitro (Savaldi-Goldsteinet al., 2000). Using site-directed mutagenesis, it

was demonstrated that the LAMMER motif isnecessary for PK12 kinase activity but not for itsinteraction with the SR proteins. PK12, as ex-pected of a Clk/Sty kinase, is localized to thenucleus. The finding that PK12 transcript andactivity is induced by ethylene suggests that thishormone may influence pre-mRNA splicing inplants through phoshporylation of SR proteins. Inaddition to three lammer-type kinases, there is anortholog of SRPK in the Arabidopsis genome(www.arabidopsis.org).

In animals, interaction among SR proteins,nuclear import of SR proteins, intranuclear move-ment of SR proteins, and splice site selectionappear to be mediated by phosphorylation of theRS domain in SR proteins (Gui et al., 1994a;Colwill et al., 1996a: Caceres, 1997; Colwill etal., 1996b; Misteli et al., 1997; Misteli andSpector, 1997; Duncan et al., 1998; Kanopka etal., 1998; Misteli, 1999; Yeakley et al., 1999).Using a coprecipitation analysis it was demon-strated that AFC2 kinase binding to one of thesubstrates (SR33) is dependent on its phospho-rylation status (Golovkin and Reddy, 1999). Inmammalian cells, overexpression of active Clk/Sty causes dissociation of nuclear speckles andredistribution of SR proteins within the nucleus(Colwill et al., 1996b). The interaction of U1-70K with plant SR proteins and interaction amongSR proteins is summarized in Figure 12. Twoplant SR proteins (SRZ21 and SRZ22) that sharesome features with animal 9G8-like protein in-teract with only the full-length U1-70K, whereasSR33 and SR45 interact with the C-terminal argi-nine-rich region of U1-70K. In addition, SR33interacts with itself and SR45 but not with SRZ21or SRZ22 proteins (Golovkin and Reddy, 1999).In animals, 9G8 splicing factor is not known tointeract with U1-70K. Furthermore, the interac-tion of U1-70K with an SR protein containingtwo RS domains with U1-70K is also novel toplants. Our data demonstrate that the plant U1-70K interacts with a novel set of SR proteins andsuggest that early stages of spliceosome assem-bly especially splice site selection in plants islikely to be different from animals. The earlyevents in the formation of plant spliceosomeassembly are shown in Figure 12. For details seethe legend of the figure.

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In animals, many pre-mRNA processing fac-tors are enriched in “speckles” that correspond tothe interchromatic granule clusters (Spector, 1996).Some SR proteins shuttle between nucleus andcytoplasm and such shuttling is affected by phos-phorylation status of their serine/arginine-richdomain (Caceras et al., 1998). Heterogeneous RNPA1 (hnRNP A1), a member of the hnRNP A/Bfamily, also shuttles continuously between thenucleus and cytoplasm (Piñol-Roma and Dreyfuss,1992). Under stress conditions, hnRNP A1 leveldecreases in the nucleus and accumulates in thecytoplasm. This redistribution is mediated byphosphorylation through the MKK3/6-p38 path-way (van der Houven van Oordt et al., 2000),suggesting a link between a signal transductionpathway and alternative splicing.

Recent studies suggest that RNAs and RNAprocessing factors move by diffusion throughinterchromatin space (Lewis and Tollervey, 2000;Misteli, 2001). Three major pre-mRNA process-ing events — capping at the 5' end, splicing andpolyadenylation at the 3' end — are coupled totranscription. This is accomplished by recruit-ment of all components necessary for these eventsto RNA polymerase II to form a transcriptosome(Gall et al., 1999). The carboxy-terminal domain(CTD) of RNA polymerase II, which consists ofmany copies of a heptad repeat (YSPTSPS) and isthe major site for phosphorylation, plays an im-portant role in coupling transcription and pre-mRNA splicing (Misteli and Spector, 1999; Lewisand Tollervey, 2000). Removal or overexpressionof CTD impairs pre-mRNA processing (Cho et

FIGURE 12. Possible roles of plant SR proteins during the early stages of spliceosomeformation prior to the formation of pre-spliceosome complex. Proteins that bind touridine-rich sequences in introns (shown as filled circles ) and serine/arginine-richproteins are likely to play an important role in intron recognition. Several SR proteinsincluding some novel ones and the proteins that bind uridine-rich sequences have beenidentified in plants. Plant U1-70K is known to interact with at least four different SRproteins. ESE, exonic splicing enhancer; SR, serine/arginine-rich protein; BBP, branchpoint binding protein; U2AF, U2 auxiliary factor.

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al., 1997; McCracken et al., 1997a; McCracken etal., 1997b).

In addition to SR proteins, in animals severalhnRNP (heterogenous ribonucleoprotein particle)proteins, among other things, are involved inmRNA processing and regulate alternative splic-ing (Krecic and Swanson, 1999). Most of thehnRNP proteins contain one or more RNA bind-ing domain and other auxiliary domains. About10 hnRNP-like proteins have been identified inthe Arabidopsis database and a few have beencharacterized experimentally (reviewed inLorkovic et al., 2000b). These include six mem-bers similar to hnRNPA/B, two related to hnRNPH/F and one hnRNPI, also called polypyrimidinetract-binding (PTB) protein. Some plant hnRNP-like proteins (RZ-1 and UBP1) have no homologsin animals (Hanano et al., 1996; Lambermon etal., 2000).

V. INTRON AND EXON DEFINITIONMODEL

The mechanisms by which splice sites in pre-mRNAs of multicellular eukaryotes are selectedwith high fidelity are largely unknown. Althoughthe short consensus sequences at the splice andbranch sites are required for splice site recognition,the information content in these sites alone is notsufficient. Accumulating evidence suggests thatother factors such as length and sequence of exonsand introns are also important in splice site selec-tion.

Two different models named exon definitionand intron definition have been proposed to illus-trate the mechanisms involved in splice site rec-ognition (Figure 13) (Robberson et al., 1990;Talerico and Berget, 1994; Berget, 1995). Ac-cording to the exon definition model, the splicingmachinery initially recognizes splice sites aroundan exon and assembles on the exon and then theneighboring exons are juxtaposed (Robberson etal., 1990; Berget, 1995) (Figure 13). In this case,the sequences in introns are not involved in splicesite recognition. This model is favored in caseswhere the introns are very large and exons arefairly small as in the case of vertebrate introns(Hawkins, 1988; Sterner et al., 1996; Consortium,

2001). According to the exon definition model,mutations in the 5' splice site should lead to skip-ping of a specific exon. Recent studies with sev-eral mutants have provided evidence in support ofthe exon definition model (Brown, 1996; Lazarovaet al., 1998; Simpson et al., 1998). Three mutantalleles of Arabidopsis COPI gene show exon skip-ping (Simpson et al., 1998). A mutation in cop1-1(G to A change at 4 nt upstream from the 3' splicesite of intron 5) and cop1-2 (the first nucleotide ofintron 6 is changed from G to A), the mutationsresult in skipping of exon 6. The mutation incop1-8 (the last nucleotide of intron 10 is changedfrom G to A) causes skipping of exon 11. Analy-sis of an intragenic suppressor of the Arabidopsisfloral organ identity mutant (ap3-1) also supportsexon definition model (Yi and Jack, 1998). Thesplicing patterns in these three mutant lines ofArabidopsis provide evidence for exon defini-tion mechanisms operating in plant splicing.Exons in animal pre-mRNAs contain purine-richexonic splicing enhancers (ESEs), which arerecognized by regulatory splicing factors (Sun etal., 1993; Tanaka et al., 1994; Tacke and Manley,1999). Although plant exons are rich in GC content,it is not known if this bias is in any way involvedeither in the splice site recognition or regulation ofsplicing (Carle-Urioste et al., 1994; Carle-Urioste etal., 1997; Latijnhouwers et al., 1999)

The intron definition model proposes thatthe splicing machinery recognizes the se-quences in the intron and assembles on theintron (Figure 13). According to this model,the sequences in the exon have little or no rolein splice site recognition. This type of mecha-nism is believed to operate in genes that havesmall introns (Talerico and Berget, 1994).However, it is becoming clear that both thesetypes of interactions occur simultaneously inrecognizing multiple splice sites. Based on theimportance of sequence elements in plant in-trons, it is thought that recognition of plantintrons involves intron definition. However,there is evidence to support both these modelsin plants (Carle-Urioste et al., 1994; Brown,1996; McCullough et al., 1996; McCulloughand Schuler, 1997; Lazarova et al., 1998;Simpson et al., 1998; Yi and Jack, 1998; Lalet al . , 1999). Mutat ional studies with

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β-conglycinin suggest that intronic U-rich se-quences function actively in 5' recognition bypromoting recognition of the 5' splice site atthe UA transition (McCullough and Schuler,1997). In vivo splicing studies using two in-trons with mutations in an intron indicatedthat the splice site selection patterns in plantnuclei are defined primarily by sequenceswithin the intron and secondarily by weak in-teractions across exons (McCullough et al.,1996; Egoavil et al., 1997). There is evidenceto suggest that sequence elements on eitherside (exonic sequences and UA-rich sequencesin the intron) of the 5' site cooperate in defin-ing the 5' splice site (Lou et al., 1993b; Egoavilet al., 1997; Schuler, 1998). Similarly, the 3'splice site is defined by the position of the UAtransition points, the U-richness preceding the3' splice site and identity of the -4 nucleotide(Lou et al., 1993a; Baynton et al., 1996; Merritt etal., 1997; Schuler, 1998). Guanine at the -4 posi-tion appears to be required for 3' splice site defi-nition (Baynton et al., 1996). The current evi-dence indicates that UA- or U-rich elementsdistributed throughout the intron define the bound-aries of the intron by promoting recognition ofupstream 5' and downstream 3' splice sites.

The second exon in most plant inver-tases is only 9 nt in length and encodes three

highly conserved amino acids in all plantinvertases. Analysis of a potato invertasepre-mRNA has shown that the upstream in-tron 1 is required for exon 2 inclusion,whereas the downstream intron 2 is not(Simpson et al., 2000). Further analysis ofintron 1 by mutagenesis resulted in identifi-cation of two sequence elements, a putativebranch point sequence, which is further up-stream from the normal position, and an ad-jacent U-rich region, necessary for exon 2inclusion. These results suggest that thesesequence elements therefore act as a splic-ing enhancer and appear to function via in-teractions between factors bound at thebranch point/U-rich region and at the 5'splice site of intron 2, activating removal ofthis intron followed by removal of intron 1.

VI. ALTERNATIVE SPLICING IN PLANTS

It is becoming apparent that alternative splic-ing of pre-mRNAs accounts for a large propor-tion of proteomic complexity in eukaryotes. Theprocess of splicing becomes even more complexin the case of some transcripts that undergo alter-native splicing where a pre-mRNA from a singlegene is processed differently to produce several

FIGURE 13. Exon and intron definition models of pre-mRNA splicing. According to the exon definition model thesplicing machinery recognizes splice sites around an exon and assembles on the exon and then the neighboringexons are juxtaposed. The intron definition model proposes that the splicing machinery recognizes the sequenceelements in the intron and assembles on the intron. Filled circles on the intron represent proteins that bind U-richsequences in the introns. Evidence in support of both these models exists in plants. ESE, exonic splicing enhancer;SR, serine/arginine-rich protein; BBP, branch point binding protein; U2AF, U2 auxiliary factor.

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functional mRNAs, each with distinct functions(Smith et al., 1989). Several factors such as thecell type, developmental stage of an organ ororganism, or signals influence the processing paththat a transcript takes (Smith and Valcarcel, 2000).For instance, sex determination in Drosophila iscontrolled by alternate splicing of genes (Smith etal., 1989; Tian and Maniatis, 1992). The produc-tion of multiple transcripts from a pre-mRNA isdue to exon skipping, alternative 5' splice site,alternative 3' splice site, activation of cryptic splicesites and/or inclusion of introns (Smith et al.,1989) (Figure 14). The combinatorial joining ofexons by alternative splicing is an elegant waythat most eukaryotes use to generate several dis-tinct proteins from a single transcript. Alternativesplicing of pre-mRNAs resulting in the produc-tion of distinct proteins is fairly common in ani-mals. For example, over 20 different isoforms offibronectin are produced from a single pre-mRNA.In animals, based on a large scale EST data analy-sis, it is estimated that pre-mRNAs from about38% of all human genes undergo alternative splic-ing, suggesting that alternative splicing contrib-utes to a large portion of proteomic complexity inmulticellular organisms (Hanke et al., 1999; Brettet al., 2000). These authors may have underesti-mated the prevalence of alternative splicing asthey examined EST alignments covering only aportion of a gene. Alignment of genes on humanchromosome 22 with available ESTs and cDNAsindicate that about 59% (145 out of 245) of thegenes are alternatively spliced to produce two ormore transcripts (Consortium, 2001). In C. elegansabout 22% of genes for which ESTs were foundshowed alternative splicing. Comparison of ge-nomic sequences with all available ArabidopsisESTs should aid in identifying what fraction ofpre-mRNA undergo alternative splicing. Althoughit is not known exactly what percentage of plantpre-mRNAs undergo alternative splicing, a largenumber of primary transcripts in plants have beenreported to produce more than one mRNA byalternative splicing (see Table 3). Alternative splic-ing of some pre-mRNAs is due to utilization ofdifferent 5’ splice sites and a common 3’ splicesite (Werneke et al., 1989; Hirose et al., 1993;Gorlach et al., 1995), whereas in others it is dueto utilization of a common 5’ splice site and dif-

ferent 3’ splice sites (Grotewold et al., 1991). Inthe case of Arabidopsis U1-70K, generation of alarge transcript is due to skipping of both 5’ and3’ splice sites of a long (910 bp) intron (Golovkinand Reddy, 1996). The functional effects of alter-native splicing on the encoded protein productscan be at the level of activity (loss, gain, or modi-fication) or cellular localization of the protein(Table 3). However, the functional significanceof alternatively spliced transcripts is known onlyin a few cases.

The rubisco activase pre-mRNA produces twotranscripts due to the usage of alternative 5' splicesite and two proteins are produced from thesetranscripts. Only one of the proteins (large form)is redox regulated (Werneke et al., 1989; Zhangand Portis, 1999; Zhang and Komatsu, 2000).Amylose content in rice is shown to be controlledby alternative splicing of Waxy pre-mRNA (Wanget al., 1995). In cultivated rice there are two wild-type alleles (Wxa and Wxb) and the expression ofWxa is tenfold higher than Wxb due to inefficientsplicing of intron 1 (Isshiki et al., 1998a). Re-cently, two mutations (du-1 and du-2) that reducethe splicing of only Wxb have been isolated, sug-gesting that DU-1 and DU-2 encode factors thatregulate splicing (Isshiki et al., 2000). The to-bacco N gene product, which confers resistanceto tobacco mosaic virus, is a member of a class ofresistance (R) genes with homology to Toll-IL-1receptor. It contains a nucleotide-binding site andleucine-rich repeats (Whitham et al., 1994). Re-cently, N gene has been shown to undergo alter-native splicing to produce two transcripts, Ns andNl. The short (Ns) and long transcripts (Nl) areproduced by splicing of an alternative exon presentin the third intron. The exon in the third intron isretained in the Nl transcript. The small transcript(Ns) encodes the full-length protein (1144 a. a.).However, the inclusion of the alternative exon inthe long transcript shifts the reading frame result-ing in a truncated protein (652 a. a) that lacks 13of the 14 leucine-rich repeat regions. Prior toinfection and during the first three hours afterTMV infection the Ns form is more prevalent,whereas the Nl form becomes more prevalentafter 4 to 8 h (Dinesh-Kumar and Baker, 2000).Transgenic plants expressing Ns transcript didnot show complete resistance to TMV. However,

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TABLE 3Some Plant pre-mRNAs That Are Known to Undergo Alternative Splicing

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TABLE 3 (continued)

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TABLE 3 (continued)

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TABLE 3 (continued)

transgenic lines containing Ns cDNA with intron3 or a genomic region capable of producing bothNs and NL transcripts showed complete resis-tance to TMV, suggesting that both transcripts arenecessary to confer TMV resistance (Dinesh-Kumar and Baker, 2000). The rice succinate de-hydrogenase subunit B pre-mRNA produces twotranscripts that encode two very different func-tional mitochondrial proteins (Kubo et al., 1999).

Several genes encoding spliceosomal proteinsor putative RNA binding proteins have been shownto undergo alternative splicing. FCA, a floweringgene that encodes a putative RNA binding pro-tein, produces four distinct transcripts by alterna-tive splicing (Macknight et al., 1997). The levelof different transcripts appears to regulate flower-ing time. SR33 produces multiple transcripts byalternative splicing. The alternatively spliced prod-ucts contain an additional exonic sequence of 163nucleotides that originates from a large (764 nucle-otides) region in intron #3 (Golovkin and Reddy,1999). Some of these transcripts differ from oth-ers in their 3’ untranslated sequences.

Analysis of the 5' region of pre-mRNA en-coding a starch-branching enzyme by RT-PCRhas shown that first two exons and introns aredifferentially processed in wheat kernel to pro-duce three variants of mRNA (Baga et al., 1999).One form contained exons 1, 2, and 3, whereasothers contained either intron 1 or missing exon 2,

resulting in N-terminal variants with different tran-sit peptides. A pre-mRNA encoding a noveldiacylgylcerol kinase in tomato has been shownto produce two functional transcripts by inclusionor exclusion of a last exon containing the codingregions for a calmodulin-binding domain (Sneddenand Blumwald, 2000). Both transcripts producefunctional kinase. Since the diacylglycerol kinasefrom the long transcript contains a calmodulin-binding domain, it is likely that the activity and/or localization of this form is regulated by cal-cium and calmodulin.

Studies with animal systems show that theexpression of SR proteins is regulated at the tran-scriptional and/or posttranscriptional level. Forexample, SRp20 is highly expressed in thymus,testis, and spleen, but not in liver or kidney of mice(Ayane et al., 1991). The transcription of some SRproteins is regulated by hormones and mitogens(Fu, 1995). In addition, several SR protein pre-mRNAs are known to undergo alternative splicingto produce multiple isoforms (Ge and Manley,1990; Fu and Maniatis, 1992; Popielarz et al., 1995).For example, alternative splicing of ASF pre-mRNAproduces three isoforms of ASF (ASF-1, ASF-2,and ASF-3) of which ASF-2 and ASF-3 isoformslack an RS domain (Ge and Manley, 1990). Mul-tiple isoforms have also been detected in other SRproteins such as SRp55, SRp40, and SC35. In thecase of SC35, alternative splicing events do not

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effect its coding region but alter the 3’ untranslatedregion (Fu, 1995). The expression of some of theSR genes in plants is also regulated by alternativesplicing (Table 3) (Lazar et al., 1995; Lopato et al.,1996b; Lopato et al., 1999b). However, in mostcases the function of alternatively spliced tran-scripts and their encoded proteins is not known.

Alternative splicing is one of the most com-plex cellular processes in eukaryotes. The resultsfrom both plants and animals indicate tissue-spe-cific and temporal regulation of alternative splic-ing. The mechanisms that regulate alternativesplicing of plant pre-mRNAs are entirely un-known. Recent studies with non-plant systemsindicate the regulation of alternative splicing isaccomplished through a combinatorial interactionof positive and negative regulators in pre-mRNAthat are recognized by the members of hnRNPand splicing factors. It is thought that splicingfactors that are essential for constitutive splicingmay also function in alternative splicing by regu-lating splice-site choice. The splice site choice islikely to be regulated by the ratio of various splic-ing factors in the spliceosome (Smith andValcarcel, 2000). In animals, there is some evi-dence to indicate that the ratio of splicing factorsmodulates alternative splicing. For example, in-creasing the ratio of SF2/ASF relative to hnRNPA1 has been shown to alter the splice site selec-tion both in vitro and in vivo (Mayeda and Krainer,1992; Caceres et al., 1994). Stress conditions suchas osmotic shock and UV radiation alter the dis-tribution of hnRNP A1 resulting in its accumula-tion in cytoplasm with a concomitant drop in thenucleus. This redistribution correlates withchanges in the alternative splicing pattern of areporter pre-mRNA, suggesting that subcellulardistribution of splicing factors influence the splicesite selection in vivo (van der Houven van Oordtet al., 2000). Apart from redistribution of splicingfactors, transcriptional regulation may also con-trol the amount of these factors. The level ofsplicing factors at the splice sites within the nucleusmay also be controlled by intranuclear compart-mentalization of splice factors. There is someevidence in animals that such intranuclear redis-tribution may also be controlled by phosphoryla-tion of splicing factors (Duncan et al., 1997;Graveley, 2000; Smith and Valcarcel, 2000).

In Arabidopsis, there are 232 proteins withRNA recognition motif (RBD and RNP) and 84with DEAD/DEAH box containing helicases(Venter et al., 2001). The number of these twogroups of proteins is slightly larger in plants whencompared with humans (Table 4). There are 224protein with RNA recognition motif and 66 pro-teins with DEAD/DEAH box proteins in humans.Although it is not clear how many of the proteinswith RRM and DEAD/DEAH are involved inpre-mRNA splicing, it is likely that several ofthese play a role in pre-mRNA splicing.

VII. EFFECT OF STRESSES ON PRE-MRNA SPLICING

There are several examples where stress sig-nals have been shown to influence pre-mRNAsplicing (reviewed in Luehrsen et al., 1994;Lorkovic et al., 2000b). For example, the ratio ofthe two transcripts (SR1B/SR1), which are gener-ated by alternative 3' splice site utilization in in-tron 9, is controlled by temperature. The ratio ofSR1B/SR1 increases in a temperature-dependentmanner, suggesting a role for SR1B in the adap-tation of plants to high temperature (Lazar andGoodman, 2000). The expression of the Bronze-2 (Bz2) gene in maize, which encodes a glu-tathione-S-transferase, is highly induced by heavymetals such as cadmium (Marrs and Walbot,1997). Treatment of maize seedlings with cad-mium caused about a 20-fold increase in Bz2message accumulation and a 50-fold increase inthe presence of the unspliced, intron-containingtranscript. However, the splicing of other testedintron-containing genes is not affected by stress,suggesting that the effect of cadmium is specificto Bz2 (Marrs and Walbot, 1997). In yeast andmetazoans splicing is inhibited by heat stress (Yostet al., 1990). In plants splicing of some pre-mRNAs(e.g., soybean hsp-26A; Bz2; petunia hsp70) isunaffected by heat stress (Czarnecka et al., 1988;Nash et al., 1990), whereas splicing of a few pre-mRNAs (maize polyubiquitin and hsp70,Arabidopsis hsp81, rice waxy gene) is partiallyinhibited by severe heat stress (Christensen et al.,1992; Hopf et al., 1992; Luehrsen et al., 1994;Larkin and Park, 1999). The mechanisms by which

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some stresses influence splicing in plants arelargely unknown.

VIII. FUNCTIONS OF INTRONS

The presence of introns adds another regula-tory step in controlling gene expression. As de-scribed above, alternative splicing results in gen-eration of structurally and functionally distinctproteins from a single gene. It is becoming moreand more evident that alternative splicing contrib-utes to the proteomic complexity of multicellulareukaryotes. Introns have been shown to play animportant role in controlling gene expression inplants (Luehrsen and Walbot, 1991; Norris et al.,1993). The expression of several genes is en-hanced by inserting introns in the cDNAs (Calliset al., 1987; Vasil et al., 1989; McElroy et al.,1990; Tanaka et al., 1990; Luehrsen and Walbot,1991; Maas et al., 1991; Christensen et al., 1992).Intron-mediated enhancement of gene expression(IME) has been discussed in previous reviews(Luehrsen et al., 1994; Simpson and Filipowicz,1996). The addition of the alcohol dehydrogenase1-S (Adh 1-S) intron 1 in the transcription unit ofmaize and reporter gene constructs has been shownto increase gene expression in cultured maizecells (Luehrsen and Walbot, 1991). Inclusion ofthe third intron of the maize actin gene also in-creased the expression of reporter genes (Luehrsenand Walbot, 1991). The inclusion of an intronresults in increased levels of steady-state RNAand also unspliced RNAs in transient assays(Luehrsen and Walbot, 1991). However, no obvi-

ous sequence similarities have been found amongintrons that enhance gene expression. The firstintron (1028 nt) in shrunken 1 has been shown toenhance its expression (Vasil et al., 1989; Maas etal., 1991). It appears that the composition of thisfragment rather than its sequence is important forenhanced gene expression. The expression of areporter gene containing the first two introns ofphosphoribosylanthranilate transferase (PAT1) isabout 30-fold greater than the reporter gene with-out the introns (Rose and Last, 1997). By analyz-ing the transcriptional rate and steady-state levelsof mRNA it was shown that these introns actposttranscriptionally to increase the steady-statelevel of mRNA (Rose and Last, 1997). In additionto IME, introns have been implicated in tissue-specific and regulated expression of certain genes.For example, plastid- and light-dependent expres-sion of spinach PsaD gene requires an intron(Bolle et al., 1996). An intron in the floral homeoticgene (AGAMOUS) has enhancer sequences for itsexpression in flowers (Busch et al., 1999). In rice,tissue-specific expression of a tubulin gene(OsTubA1) is mediated by intron 1 (Jeon et al.,2000).

There are instances where the introns encodeproteins or inclusion of an intron by alternativesplicing produces very different proteins (Kubo etal., 1999; Yu et al., 1999). In mammals, the genesthat encode U14 through U40 (e.g., U14-U21,U23, U24, U32-U40) are within the introns ofprotein coding region of other genes. These in-trons once excised from the pre-mRNA are pro-cessed to generate snoRNAs. It has been pro-posed that the presence of introns permits shuffling

TABLE 4Proteins with DEAD/DEAH box or RRM (RNa Recognition Motif) in Arabidopsis andAnimals Whose Genomes Have Been Sequenced (Compiled from Data Presented inVenter et al., 2001)

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of exons to generate novel genes with new com-binations of functional domains (Gilbert et al.,1997). In addition, introns are implicated in en-hancing within the gene recombination and there-fore increase selection efficiency (Gilbert et al.,1997; Duret, 2000).

IX. CONCLUSIONS

Although several in vivo pre-mRNA splicingstudies in plant cells have led to identification ofsome of the cis-elements necessary for efficientand accurate splicing of plant pre-mRNAs, ourunderstanding of the mechanisms that are involvedin recognition of introns and exons, and regulationof splicing in plants is still very rudimentary. In-creasing evidence indicates the plant introns havesome unique features and the mechanisms of in-tron recognition in plants are likely to be different,at least in some aspects, from yeast and animals.The lack of a plant derived in vitro splicing systemrequires that in vivo methods be used to identifyspliceosomal proteins and interaction among them.Use of the yeast two- and three-hybrid systems(Drees, 1999; Kraemer et al., 2000; Zhang et al.,2000) should enable identification of interactingprotein partners of plant spliceosomal proteins. Inaddition, with new technologies researchers arenow in the exciting position of being able to studythe dynamics of various splicing proteins in thenucleoplasm of live cells using fluorescent report-ers. Fluorescence resonance energy transfer (FRET)(Tsien and Tsien, 1990; Llopis et al., 2000) andfluorescence recovery after photobleaching (FRAP)(Phair and Misteli, 2000; Misteli, 2001) will permitanalysis of interaction of various proteins impli-cated in spliceosome formation and their dynamicsin vivo. Use of such technology with animal cellsis already providing some unexpected interestinginsights into the organization and dynamics of splic-ing factors within the nucleus. Inactivation of pu-tative splicing proteins, identified based on se-quence/domain similarity searches, by reversegenetics and RNAi (RNA interference) shouldprovide insights into the function of at least someof them. In the near future we should have betterunderstanding of the contribution of alternativesplicing in generating proteomic complexity and in

controlling various aspects of plant growth anddevelopment. Identification of cis- and trans-act-ing factors that are involved in recognition of plantintrons is critical to the understanding of pre-mRNAsplicing in plants. Such information would be veryuseful for optimal expression of foreign genes intransgenic plants. A few reports have shown thatan introduced transgene in sense orientation caninterfere with splicing of pre-mRNAs from theendogenous as well as the introduced gene (Mishraand Handa, 1998; Metzlaff et al., 2000)

Numerous studies on pre-mRNA splicing inplants suggest that there are likely some differ-ences between plants and animals. The increasingrealization that alternative splicing contributesconsiderably to proteomic complexity and that itplays an important role in growth and develop-ment of higher eukaryotes is likely to make thestudy of splicing more prominent in the near fu-ture. Despite the growing realization of the im-portance of alternative splicing in increasing thecoding capacity of genes, virtually nothing isknown about the mechanisms that regulate alter-native splicing in plants. There are a large numberof proteins with RNA recognition motif (RRMand RNP), serine/arginine-rich proteins andDEAD/DEAH box helicases in the Arabidopsisdatabase (Venter et al., 2001). The presence of anapproximately equal number of these families ofproteins in plants and humans suggests that thepre-mRNA splicing in plants is likely to be ascomplex as in humans. Several studies using syn-thetic or chimeric constructs have shown that splicesite selection and splicing efficiency of plant pre-mRNAs depend, in addition to splice site se-quences, strongly on compositional differencebetween exons and introns.

It is likely that at least some sequence ele-ments important for proper splicing of the nuclearprecursor to mRNAs are conserved during evolu-tion. With the availability of the complete ge-nome sequence of several phylogenetically di-verse organisms and the expected completion ofgenomes of crop plants (Eckardt, 2000; Gai et al.,2000; Initiative, 2000; Sasaki and Burr, 2000;Consortium, 2001), it should be possible to usebioinformatics tools to identify sequences in theintrons and exons that are critical for accuratesplicing. Furthermore, such analyses should also

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help in identifying the sequence elements thatinfluence splice site selection. The activity of suchpredicted elements can then be tested experimen-tally. Sequence analyses studies have already ledto the discovery of a second pre-mRNA splicingpathway. The next challenge will be to identifyproteins that recognize the signal and initiatespliceosome formation. Genomics will lead tomore surprises in this and other fields in the nearfuture.

ACKNOWLEDGMENTS

My special thanks to Dr. Maxim Golovkinfor his contributions to splicing research in mylaboratory. I would like to thank Drs. MaximGolovkin, Irene Day and Vaka Reddy for criti-cally reading the manuscript; Maxim Golovkin,Bryan Criswell and Vaka Reddy for their help inpreparing the figures. Pre-mRNA splicing researchin my laboratory is supported by grants fromDepartment of Energy, USDA NRICGP, Colo-rado RNA Center and Agricultural ExperimentStation.

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