8
452 RNA-binding proteins, which are involved in the synthesis, processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellular regulatory proteins. Although relatively few RNA-binding proteins have been studied in plants, they are being identified with increasing frequency, both genetically and biochemically. RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have been elegantly analyzed in Clamydomonas reinhardtii have counterparts with similar functions in higher plants. Several recent reports describe mutations in genes encoding RNA-binding proteins that affect plant development and hormone signaling. Address Biology Department and Life Sciences Consortium, 519 Wartik Laboratory, Pennsylvania State University, University Park, Pennsylvania 16802, USA Current Opinion in Plant Biology 2002, 5:452–459 1369-5266/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. Published online 30 July 2002 Abbreviations ABA abscisic acid abi5 abscisic acid insensitive5 abh1 abscisic acid hypersensitive1 ARE AU-rich element bcd bicoid CBC cap-binding complex ds double-stranded eIF translation initiation factor Hsp27 Heat shock protein27 hyl1 hyponastic leaves1 MAPK mitogen-activated protein kinase NLS nuclear localization signal osk oskar PABP poly(A)-binding protein PP2C protein phosphatase 2C RB RNA-binding RBD RNA-binding domain RNP ribonucleoprotein particle sad1 supersensitive to ABA and drought1 SAUR small auxin-upregulated snRNP small nuclear RNP STAU Staufen UTR untranslated region Introduction RNA-binding proteins have received less attention in plants than in other eukaryotes. Perhaps the most exten- sively studied are the proteins involved in the redox regulation of chloroplast mRNA translation and stability [1]. But plant RNA-binding proteins are beginning to surface with increasing frequency. The Arabidopsis caf and hua1 floral development mutations are both in genes encoding RNA-binding proteins [2,3]. Several recent reports implicate RNA-binding proteins in hormone signaling [4–6], and these proteins appear to be involved in circadian rhythms in plants, as they are in other organisms [7]. Anticipating that many more RNA-binding proteins will soon come to light through biochemical and genetic experiments, I briefly explore how these proteins recognize their substrates. I then describe several plant RNA-binding proteins in the context of the regulatory mechanisms in which they are known or likely to participate. Proteins accompany RNA molecules from cradle to grave. In eukaryotes, RNA-binding proteins participate in synthesizing, processing, editing, modifying and exporting RNA molecules from the nucleus [8,9 ,10 ]. They carry RNA molecules between cells and to their destinations within cells [11 ]. They are involved in all aspects of translating mRNAs, as well as in storing them while they are not being translated [12 ,13]. RNA-binding proteins regulate the stability of mRNA and degrade it [14,15 –17 ]. In addition to their involvement in these very central aspects of decoding the information stored in DNA, referred to as ‘RNA metabolism’, they are also involved in certain aspects of chromosome structuring and coarse regulation, such as telomere maintenance [18] and X-chromosome inactivation [19]. Some RNA-binding proteins are old and some are new Many proteins of RNA metabolism are ancient, their origin predating the divergence of bacteria, archaea and eukaryotes. Indeed, they may reflect the conjectured transition from an RNA-based to a protein-based form of life. A recent phylogenetic study reported that almost half of the approximately 100 domains that are common to the proteins involved in RNA metabolism probably date to the last universal common ancestor of the major living kingdoms [20]. These conserved protein ‘motifs’ identify both non-catalytic domains, primarily RNA-binding domains (RBDs), and catalytic domains that are involved in RNA modification (e.g. thiolation, isomerization and deamination), ATP or GTP hydrolysis (e.g. translation factors, RNA helicases and aminoacyl-tRNA synthetases), or RNA degradation (ribonucleases). About 50 major superfamilies of non-catalytic RBDs have been recognized [20]. Some are specific to certain functions, with the most ancient and highly conserved being associated with trans- lation and RNA modification. Others might be regarded as mobile or ‘promiscuous’ in evolutionary terms because they appear in proteins together with many other kinds of functional domains [20]. Yet others have arisen more recently, again in conjunction with a variety of other catalytic and interaction domains. Indeed, similar RBDs appear in proteins with an almost bewildering array of different functions [21]. Hence, the presence of an RBD is a poor predictor of what a protein does. RNA-binding proteins in plants: the tip of an iceberg? Nina V Fedoroff

RNA-binding proteins in plants: the tip of an iceberg?

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RNA-binding proteins, which are involved in the synthesis,processing, transport, translation, and degradation of RNA, are emerging as important, often multifunctional, cellularregulatory proteins. Although relatively few RNA-bindingproteins have been studied in plants, they are being identifiedwith increasing frequency, both genetically and biochemically.RNA-binding proteins that regulate chloroplast mRNA stability and translation in response to light and that have beenelegantly analyzed in Clamydomonas reinhardtii havecounterparts with similar functions in higher plants. Severalrecent reports describe mutations in genes encodingRNA-binding proteins that affect plant development andhormone signaling.

AddressBiology Department and Life Sciences Consortium, 519 WartikLaboratory, Pennsylvania State University, University Park,Pennsylvania 16802, USA

Current Opinion in Plant Biology 2002, 5:452–459

1369-5266/02/$ — see front matter© 2002 Elsevier Science Ltd. All rights reserved.

Published online 30 July 2002

AbbreviationsABA abscisic acidabi5 abscisic acid insensitive5abh1 abscisic acid hypersensitive1ARE AU-rich elementbcd bicoidCBC cap-binding complexds double-strandedeIF translation initiation factorHsp27 Heat shock protein27hyl1 hyponastic leaves1MAPK mitogen-activated protein kinaseNLS nuclear localization signalosk oskarPABP poly(A)-binding protein PP2C protein phosphatase 2CRB RNA-bindingRBD RNA-binding domainRNP ribonucleoprotein particlesad1 supersensitive to ABA and drought1 SAUR small auxin-upregulatedsnRNP small nuclear RNP STAU StaufenUTR untranslated region

IntroductionRNA-binding proteins have received less attention inplants than in other eukaryotes. Perhaps the most exten-sively studied are the proteins involved in the redoxregulation of chloroplast mRNA translation and stability [1].But plant RNA-binding proteins are beginning to surfacewith increasing frequency. The Arabidopsis caf and hua1floral development mutations are both in genes encodingRNA-binding proteins [2,3]. Several recent reports implicateRNA-binding proteins in hormone signaling [4–6], and

these proteins appear to be involved in circadian rhythmsin plants, as they are in other organisms [7]. Anticipatingthat many more RNA-binding proteins will soon come to light through biochemical and genetic experiments, I briefly explore how these proteins recognize their substrates.I then describe several plant RNA-binding proteins in thecontext of the regulatory mechanisms in which they areknown or likely to participate.

Proteins accompany RNA molecules from cradle to grave. In eukaryotes, RNA-binding proteins participate insynthesizing, processing, editing, modifying and exportingRNA molecules from the nucleus [8,9•,10•]. They carryRNA molecules between cells and to their destinationswithin cells [11•]. They are involved in all aspects oftranslating mRNAs, as well as in storing them while theyare not being translated [12•,13]. RNA-binding proteinsregulate the stability of mRNA and degrade it [14,15•–17•].In addition to their involvement in these very centralaspects of decoding the information stored in DNA,referred to as ‘RNA metabolism’, they are also involved incertain aspects of chromosome structuring and coarseregulation, such as telomere maintenance [18] and X-chromosome inactivation [19].

Some RNA-binding proteins are old and someare newMany proteins of RNA metabolism are ancient, theirorigin predating the divergence of bacteria, archaea andeukaryotes. Indeed, they may reflect the conjecturedtransition from an RNA-based to a protein-based form oflife. A recent phylogenetic study reported that almost halfof the approximately 100 domains that are common tothe proteins involved in RNA metabolism probably date tothe last universal common ancestor of the major livingkingdoms [20]. These conserved protein ‘motifs’ identifyboth non-catalytic domains, primarily RNA-bindingdomains (RBDs), and catalytic domains that are involvedin RNA modification (e.g. thiolation, isomerization anddeamination), ATP or GTP hydrolysis (e.g. translationfactors, RNA helicases and aminoacyl-tRNA synthetases),or RNA degradation (ribonucleases). About 50 majorsuperfamilies of non-catalytic RBDs have been recognized[20]. Some are specific to certain functions, with the mostancient and highly conserved being associated with trans-lation and RNA modification. Others might be regarded asmobile or ‘promiscuous’ in evolutionary terms becausethey appear in proteins together with many other kinds offunctional domains [20]. Yet others have arisen morerecently, again in conjunction with a variety of othercatalytic and interaction domains. Indeed, similar RBDsappear in proteins with an almost bewildering array ofdifferent functions [21]. Hence, the presence of an RBD isa poor predictor of what a protein does.

RNA-binding proteins in plants: the tip of an iceberg?Nina V Fedoroff

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RNA-binding proteins in plants: the tip of an iceberg? Fedoroff 453

How RNA-binding proteins do not interactwith RNAThe major categories of eukaryotic RBDs are designatedthe RNA-recognition motif (RRM), the double-stranded(ds)RBD and the K-homology (KH) domain, the latter ofwhich was first identified in the human ribonucleoproteinparticle (RNP) K protein [22,23]. The ability to discriminatebetween ds and single-stranded (ss) RNAs is important forsome proteins, such as the mammalian dsRNA-dependentprotein kinase R, a central player in the defense againstRNA viruses [24]. But the structural distinction betweends and ss forms is much more ambiguous for RNA than forDNA because cellular RNA molecules generally functionwithout their complements, and are therefore free to adoptsecondary and tertiary structures by intra-molecular basepairing. So the distinction between ds- and ssRNA-bindingmotifs is also not a good predictor of protein function. Andalthough the minimal contiguous length of dsRNA requiredfor binding in vitro appears to be 12–16 base pairs, in vivosubstrates do not necessarily conform to this minimum[23,25,26]. Worse yet, most RNA-binding proteins do notshow sequence-specific binding in vitro, although short,characteristic sequence motifs have been identified in targetRNAs for certain proteins, such as the AU-rich elements(AREs) in the 3′ untranslated regions (3′UTRs) of certainrapidly degraded mRNAs [14].

On the surface, this would not appear to be a promisingfoundation on which to base regulatory subtlety. And yetRNA-binding proteins are proving to be at the heart of thecell’s regulatory fine-tuning and integration. RNA-bindingproteins are instrumental in the asymmetric distribution ofproteins within cells [11•]. This is an essential aspect of thefunctioning of highly specialized cells, such as neurons[27], and is at the heart of the mechanisms that underliethe development of a multicellular organism from a singlecell [11•]. Many RNA-binding proteins are also turning outto have multiple regulatory functions, sometimes in differentcellular compartments [28••], providing links betweendifferent aspects of RNA metabolism.

How RNA-binding proteins recognize theirsubstrates: the case of the Staufen proteinDespite their indifferent performance in the test tube,RNA-binding proteins exhibit moderate to exquisitesubstrate specificity in vivo and may even devote different RNA-binding domains to different functions. TheDrosophila Staufen (STAU) protein provides an exception-ally well-analyzed example. Two mRNAs that bind toSTAU, bicoid (bcd) and oskar (osk) mRNAs, determine anterior–posterior polarity by localizing to and being translated at opposite poles of the Drosophila egg [29,30].They establish ‘morphogen’ gradients of the transcriptionfactors required to express the zygotic genes that deter-mine the overall body plan after fertilization.

The localization process is surprisingly complex. Itrequires that several RNA-binding proteins move the

inactive mRNAs out of the nurse cells into the egg and tothe appropriate pole by specific interactions with kinesin Iand dynein motor proteins in microtubules [31,32••].STAU is a dsRNA-binding protein that is required for thelocalization and anchoring of osk mRNA at the posteriorpole, for anchoring the bcd RNA at the anterior pole afteregg activation and, later, for the actin-dependent basallocalization of prospero mRNA during the asymmetricdivisions in embryonic neuroblasts [29,33]. The osk andbcd mRNAs are transported to their destinations in atranslationally repressed state and must be translationallyactivated at the right time and place for normal development,a process in which STAU also participates [34,35].

STAU contains five dsRBDs that are involved in differentfunctions through different mechanisms. dsRBD2 anddsRBD3 are required for the localization of both bcd andosk mRNA [26,36••]. dsRBD5 is required for the localizationof prospero, bcd and osk mRNA, as well as for the transla-tional derepression of osk mRNA and, possibly, thetranslation of bcd mRNA [33,34,36••]. dsRBD5 interactswith Miranda protein in the localization of prosperomRNA, suggesting that it functions as a protein interactiondomain [33]. Sequences in both the 3′- and 5′UTRs of oskmRNA are required for translational derepression, whichis an active process that must overcome translationalrepression by the Bruno protein [34].

The sequence elements necessary for STAU-mediatedlocalization of bcd mRNA are within a 625-bp sequence ofthe mRNA’s 3′UTR that can be folded into three longintramolecular hairpins, each stabilized by several short RNAduplexes. The importance of the predicted structures of the bcdmRNA was tested in vivo by introducing complementarity-disrupting and compensating complementarity-restoringmutations [25]. The structure, but not the precise sequence,of the intra-molecular hairpins is essential for STAU–bcd-mRNA complex formation [25]. These studies alsoproduced the surprising insight that a complementary inter-action between single-stranded loops in two different bcdmRNA molecules is required for STAU–mRNA complexformation [25]. Such loop–loop interactions, called ‘kissing,’are the first step in the formation of the extremely stableinter-molecular RNA associations that are necessary for theformation of the STAU–bcd-mRNA complex [37].

Themes in RNA–protein interactions: multiplefunctions and multiple interactionsI have recounted the STAU protein story because itillustrates several of the common themes in the biology andbiochemistry of RNA-binding proteins. First, RNA-bindingproteins commonly have more than one function and oftenlink two separate processes, such as intracellular localizationand translation in the case of STAU. Second, the RBDs ofRNA-binding proteins mediate both protein–nucleic-acidand protein–protein interactions. Third, RNA–proteininteractions are very specific in vivo. Although STAU isknown to interact with more than one mRNA, it does not

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interact with many and its specificity results from its abilityto recognize a structure that contains both intramolecularand intermolecular RNA duplexes.

RNA-binding proteins in plantsIn view of the fact that the Arabidopsis genome encodesmore than 200 putative RNA-binding proteins [38], it isperhaps surprising that so few have so far been identifiedthrough either biochemical experiments or the analysis ofmutations. We have much less information about whatRNA-binding proteins do in plants than in other eukary-otes. However, as noted earlier, plant RNA-bindingproteins are beginning to surface with increasing frequen-cy, as are experimental indications that other such proteinswill be discovered. In the remainder of this review, I coverthe well-studied RNA-binding proteins that regulate thetranslation of chloroplast mRNAs, and discuss othersightings, putting them in the context of the regulatorymechanisms known in other organisms.

RNA-binding proteins mediate the light-regulated translation of chloroplast mRNAsLight regulates the supply of certain chloroplast proteins,including the chlorophyll apoproteins of photosystems I

and II (encoded by the chloroplast psaA and psaB genes)and the D1 protein of photosystem II (encoded by thechloroplast psbA gene). This light regulation is primarilypost-transcriptional and utilizes RNA-binding proteinsthat are encoded in the nuclear genome [39–41]. Thetranslation of psbA mRNA, a particularly well-studiedexample, is activated by the assembly on its 5′UTR ofan RNA-binding complex comprising four proteins, designated RB38, RB47, RB55, and RB60 (Figure 1; [41]).RB47 interacts directly and specifically with the 5′UTRand is required for the initiation of translation [42]. Thestability of the mRNA also depends on the ability ofribosomes to associate with the mRNA–protein complex[42–44]. RB47 is a member of the eukaryotic poly(A)-binding protein (PABP) family and is targeted to thechloroplast, where it associates with thylakoid mem-branes [42,45]. Although PABPs are known to inhibitRNA degradation and stimulate translation in eukaryotes[46], the translational machinery of the chloroplast iscloser to those of prokaryotes and transcripts are generallynot polyadenylated. Instead, translation is controlled bythe imported RB47 eukaryotic RNA-binding proteinthrough a highly specific interaction with the mRNA’sA-rich 5′UTR.

Figure 1

S SH HS S

H H S SS S

S SH H

RB55

Dark

PSIIPSI

Light

ADP

RB47

5′ UTR 3′ UTR

5′ UTR 3′ UTR

PSIIPSI

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RB55

Thioredoxinreductase

S S

RB55

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P

S S

TRX

H H

Fd

RB38

RB38

RB38

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RB47

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TRX

RB60

RB60

P

psbA mRNA

psbA mRNA

psbA mRNA

••••••

KinasePhosphatase

PSI

PQ

Current Opinion in Plant Biology

A diagrammatic representation of molecular interactions in the light-regulated translation of psbA mRNA in chloroplasts. Fd, ferredoxin; PQ, plastoquinone; PSI, PSII, photosystem I and II; TRX, thioredoxin.

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RNA-binding proteins in plants: the tip of an iceberg? Fedoroff 455

RB60 regulates the assembly and disassembly of theRNA-binding protein complex. It is encoded in thenuclear genome and resembles an endoplasmic reticularprotein disulfide isomerase [47••]. RB60 is targeted tochloroplasts and partitions between the stroma and thethylakoid membranes, where it may serve to localizetranslation [47••]. The RNA-binding protein complexdissociates upon phosphorylation of RB60 by an ADP-dependent kinase [48]. Such regulation by ADP isconsistent with its accumulation in the dark [49]. Assemblyof the complex is also regulated by the reduction andoxidation of vicinal disulfide groups in RB60 [47••,50].The pool of RB60 becomes reduced with increasing light exposure, stimulating translation [47••]. Signals are transmitted to RB60 through components of photosystems Iand II. One signal is initiated by the reduction of the plastoquinone pool and a second by a reductive

signal from photosystem I, which is transduced by the ferredoxin–thioredoxin system [51]. The reduced form of plastoquinone regulates the phosphorylation of photo-system II proteins and may regulate the translation of thepsbA mRNA through a plastoquinone-activated kinase [51,52].

This well-studied regulatory system illustrates the abilityof RNA-binding proteins to fine-tune protein expressionlevels in response to external signals through both phos-phorylation changes and the reduction and oxidation ofregulatory disulfide bridges within proteins. Indeed, the twosignaling mechanisms may sense different environmentalchanges to optimally adjust the supply of the psbA-encodedD1 protein to ambient conditions [51]. Although much of thework on the psbA RNA-binding protein complex was donein Chlamydomonas reinhardtii, the same regulatory mechanismexists in Arabidopsis chloroplasts [53]. RNA-binding

Figure 2

β α

CBP20

eIF4E

eIF4E

eIF4E

eIF4E

Ribosome

ABH1?

AAAAAAA

4E-TNLS

U6 snRNP

AAAAAAA

CBP20

CBP20Ran

GTP

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Translation

?

Nuclearmembrane

Nucleus

Cytoplasm

PABP eIF4G

eIF4G

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eIF4G

AAAAAAA

eIF4A7mGpppG

5′→3′ RNAse3′→5′ RNAse

Dcp1

AAAA

A

PARN

SAD1?

AAAAAAA

Processing

RBP

RBP

RBP

HYL1?

Proteasome

Storage, regulation

orP

Ub

Ub

SAD1?

ABH1?

HYL1?

Degradation

568

23 4

7

56

23 4

71

CBP80

CBP80

CBP80

4E-TNLS

eIF4E

β

β

αα

eIF3

eIF4G

7mGpppG

7mGpppG

7mGpppG

7mGpppG

Other snRNPs

Current Opinion in Plant Biology

A diagrammatic representation of RNA-binding proteins that areinvolved in processing, nuclear export, translation and degradation ofmRNA. Ran is a GTPase that is involved in nuclear trafficking ofproteins. α and β are subunits of the importin complex. 4E-T is a protein

involved in nuclear import of eIF4E. CBP, cap-binding protein; Dcp1,cap-specific endonuclease; PARN, poly (A)-specific exoribonuclease;RBP, RNA-binding protein; snRNP, small nuclear ribonucleoproteinparticle; Ub, ubiquitin.

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proteins are also involved in regulating mRNA stabilityin plants. As it does for translation, light regulates thestability of several chloroplast mRNAs through redoxstate [54]. Nac2 and Mbb1 are C. reinhardtii proteins thatinteract specifically with the respective 5′UTRs of psbD andpsbB mRNAs to stabilize them [55]. They are structurallysimilar, internally repetitive proteins that are encoded inthe nuclear genome and imported into chloroplastswhere they are components of large non-polysomal RNPcomplexes [56,57].

Light regulates the stability of certain cytoplasmic mRNAs,illustrating the fact that this regulatory mechanism is notconfined to chloroplasts [58]. Many other plant mRNAsare known to be regulated at the level of stability [59].Although some work has been done to identify the RNAsequences responsible for the instability [60], little isknown about the role of proteins. The most extensivelycharacterized are the 3′UTR sequences, termed downstream(DST) elements, found in small RNA molecules that arerapidly upregulated by exogenous auxin (i.e. small auxin-upregulated [SAUR] proteins). SAUR RNAs turn overwith half-lives of 10–50 minutes. The analysis of tworecently isolated Arabidopsis mutations that increase thestability of SAUR transcripts is likely to shed light on theproteins involved in their rapid turnover [61].

RNA-binding proteins in hormone signalingThree recent reports describe the abscisic acid hypersensitive1(abh1), supersensitive to ABA and drought1 (sad1), andhyponastic leaves1 (hyl1) mutations, all of which renderseeds hypersensitive to germination inhibition by abscisicacid (ABA) [4–6]. The other phenotypic effects of thesemutations differ. The sad1 and hyl1 mutants showenhanced rates of water loss from leaves, implying reducedstomatal closure rates, whereas the abh1 mutant showsenhanced stomatal closure. The abh1 mutation appears tobe quite specific for ABA perception, resulting in unalteredresponses to exogenous auxin, cytokinin, methyl jasmonate,epibrassinosteroid, an ethylene precursor and an inhibitorof gibberellic acid biosynthesis [5]. The hyl1 mutant ishyposensitive to cytokinin and auxin, and exhibits suchauxin-related phenotypes as reduced root gravitropism andreduced apical dominance. It is hypersensitive to glucose,NaCl, and osmotic stress, as well as to ABA, but has normalresponses to other hormones [4]. The sad1 mutant ishypersensitive to NaCl and mannitol, in addition to itsdrought and ABA sensitivity [6].

The proteins encoded by the Arabidopsis ABH1 and SAD1genes are homologous to highly conserved eukaryoticRNA-binding proteins, providing some clues about theirfunction. The ABH1 gene encodes the Arabidopsis homologof CB80, one of the two proteins comprising the eukaryoticnuclear cap-binding complex (CBC) [62,63]. Figure 2shows a simplified scheme of mRNA metabolism, omittingtranscription for simplicity although it is known to betightly coupled to other aspects of mRNA metabolism [9•].

The CBC assembles during transcription, as soon as the5′ end of the nascent mRNA has been capped [9•]. It isinvolved in splicing and accompanies the mRNA out of thenucleus as part of an RNP complex. In yeast, translationinitiation factor 4E (eIF4E) associates with the protein 4E-T,which contains a nuclear localization signal (NLS), and istransported into the nucleus by the αβ importin complex[64]. Dissociation of the complex is catalyzed by theRanGTPase and the transport protein is recycled to thecytoplasm. eIF4G is also present in the nucleus, associateswith the CBC and may be exported with it [65].

The CBC persists after nuclear export and may beinvolved in translation initiation, but its cap-bindingfunctions are at some point taken over by eIF4E andeIF4G, which attracts eIF4A, an RNA helicase that isrequired to melt the translation-inhibiting secondarystructure of the 5′UTR [66]. The eIF4G further interactswith PABP, eIF3, and additional proteins, as well as withthe 40S ribosomal subunit, in the initiation of translation[66]. The proteins of the CBC are recycled to the nucleusby the importin system. The observed decreases in thesteady-state levels of several transcripts in the abh1mutant are consistent with the participation of the CBC inRNA processing and export from the nucleus. It is rathersurprising, however, that only 18 of 8000 genes analyzedshowed significantly lower steady-state levels of expressionin abh1 mutants than in wildtype plants, despite the factthat Arabidopsis CAP-BINDING PROTEIN80 (CBP80)is encoded by a single-copy gene [5,63]. This may bebecause other proteins compensate functionally for theloss of ABH1 or because ABH1 has an as yet unrecognizedsequence specificity and is required for the production ofjust a few mRNAs. In addition, ABH1 might have a secondfunction that is specific for transcripts of the affectedgenes. It is also important to recognize that the pheno-typic effects of the abh1 mutation may result from theabsence of just a few, perhaps even just one, protein. Forexample, transcripts of an AtPP2C gene, which encodes aprotein phosphatase 2C that may be involved in theregulation of ABA response [67], are much less abundantin abh1 plants than in wildtype plants [5].

The SAD1 protein is an ortholog of the yeast Lsm5 protein,which was originally identified as a subunit of the U6 smallnuclear RNP (snRNP) complex, which is involved innuclear RNA processing [68•]. More recently, Lsm5 wasshown to be part of a second, similar complex that is necessary for mRNA degradation. The cap-binding proteins and PABP protect mRNA from destruction, whichis effected primarily by 3′→5′ and 5′→3′ exoribonucleasesand a poly(A)-specific exoribonuclease (PARN) (Figure 2).Degradation commences with removal of the poly(A) tailand the m7GpppG cap. A heptameric protein complexcomprising six of the seven Lsm proteins present in theU6 snRNP, but containing a different member of the Lsmfamily (Lsm1 instead of Lsm8) has been identified as partof the degradative machinery [68•]. The complex associates

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with exoribonucleases and appears to be necessary fordecapping. The sad1 mutant shows defects associated withboth the production and the perception of ABA [6]. It wasselected for overexpression of a reporter gene from anABA-responsive promoter and the corresponding gene ismarkedly overexpressed, consistent with a defect in RNAdegradation. However, several other genes are expressedat lower steady-state levels in the sad1 mutant than inwildtype plants. These genes includes an AtPP2C gene, a homolog of the gene whose expression is reduced in the abh1 mutant [5,6]. If the SAD1 protein’s homology to the Lsm5 protein corresponds to a functional identity, thecomplex molecular phenotype of sad1 may reflect the dualinvolvement of SAD1 protein in both processing and mRNAstability. As with the abh1 mutation, however, the steady-state levels of rather few transcripts differ between sad1 andwildtype plants, suggesting the possibility that this proteinfunctions in the metabolism of a small subset of mRNAs.

Outside of its two dsRBDs, the HYL1 protein shows nostrong homology to the highly conserved proteins that areinvolved in RNA metabolism [4]. It contains two NLSsequences and its carboxyl terminus contains six almostperfect repeats of a 28-amino-acid sequence. This featureresembles the multiple 34-amino-acid sequence repeats inthe Chlamydomonas Nac2 and Mbb1 proteins, which stabi-lize chloroplast transcripts [56,57]. The hyl1 mutation, likethe sad1 and abh1 mutations, affects a relatively smallnumber of genes, most of which are stress- and defense-response genes. The transcript levels for most of thesegenes are higher in the hy1 mutant than in wildtype plants(C Lu, N Fedoroff, unpublished data). Among these arethe AtMPK3 and ANP1 genes, which encode a stress-activated mitogen-activated protein kinase (MAPK) and aMAPK kinase kinase, respectively [69].

Recent insights into the mechanism underlying ABAinhibition of germination provide a hint that these threemutants might have one defect in common. The ABI5 gene,identified through the ABA-insensitive abscisic acid insensitive5((abi5) mutation, encodes a transcription factor that is abundantin developing embryos but disappears after germination[70,71••]. ABA exposure during germination activates theaccumulation of ABI5 and arrests further growth [71••].Overexpression of transcription factors ABF3 (for ABA-responsive element [ABRE] binding factor3), ABF4, orABI5 in transgenic plants renders them hypersensitive toABA [71••,72], as does overexpression of the AtMPK3 gene(A Guevara-Garcia, C Lu, N Fedoroff, unpublished data).ABI5 accumulation is triggered by a much lower concen-tration of ABA in the hyl1 mutant, which exhibits elevatedlevels of activated MAPKs, than in wildtype plants(L Lopez-Molina, C Lu, N Fedoroff, unpublished data). Itmay be that the abh1 and sad1 mutants are ABA hyper-sensitive because the mutations interfere with expressionof AtPP2C genes that modulate ABA signaling, whereasthe hyl1 mutant is hypersensitive because it overexpressescomponents of the ABA-responsive MAPK cascade.

There is also preliminary evidence that the hyl1 mutationaffects protein and mRNA stability, in addition to its effecton mRNA levels (C Lu, N Fedoroff, unpublished data).Connections between protein and mRNA stability arebeginning to be investigated in other systems [73,74]. Inmammalian cells, for example, eIF4E is dephosphorylatedand eIF4G is sequestered in insoluble complexes withHsp27 during heat shock, thereby halting cap-dependenttranslation [74]. Cap-independent ARE-containingmRNAs, including the Hsp70 mRNA, are stabilizedduring heat shock and translated efficiently. AUF1, anRNA-binding protein that interacts with ARE elements todestabilize mRNAs, is sequestered in the nucleus andperinuclear region in a complex containing Hsp70, eIF4Gand PABP during heat shock, thereby stabilizing mRNAs[73]. MG132, a proteasome inhibitor, strongly stimulatesthe translation of Hsp70 through the formation of the samecomplex to stabilize mRNAs. Under normal conditions,AUF1 is ubiquitinated and degraded by the proteasome,establishing a link between mRNA stability and proteindegradation. Although the involvement of unstable proteinsin the regulation of auxin responses is well established, thepotential involvement of RNA-binding proteins in plantmRNA turnover in response to phytohormones remains tobe studied [75].

ConclusionsRecent work on eukaryotic RNA-binding proteins hasbegun to reveal the many subtle, often multiple ways inwhich they can regulate the basic cellular processes ofsynthesizing, transporting, translating and degradingmRNAs. Although less work has been done in plants thanin other eukaryotic systems, studies on chloroplastRNA-binding proteins have documented their extraordinarycapacity to closely regulate the supply of critical photo-system proteins in response to changes in light level. Theabundance of Arabidopsis genes encoding RNA-bindingproteins, and the recent identification of developmentaland hormone-response mutations in Arabidopsis genes thatencode RNA-binding proteins, suggests that such proteinswill also emerge as important players in plant morphogenesisand cellular regulation.

AcknowledgementsI would like to thank A Danon, J Schroeder and J Zhu for critical reading ofthis manuscript.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Rochaix JD: Posttranscriptional control of chloroplast geneexpression. From RNA to photosynthetic complex. Plant Physiol2001, 125:142-144.

2. Jacobsen SE, Running MP, Meyerowitz EM: Disruption of an RNAhelicase/RNAse III gene in Arabidopsis causes unregulated celldivision in floral meristems. Development 1999, 126:5231-5243.

3. Li J, Jia D, Chen X: HUA1, a regulator of stamen and carpelidentities in Arabidopsis, codes for a nuclear RNA binding protein.Plant Cell 2001, 13:2269-2281.

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4. Lu C, Fedoroff N: A mutation in the Arabidopsis HYL1 geneencoding a dsRNA binding protein affects responses to abscisicacid, auxin and cytokinin. Plant Cell 2000, 12:2351-2366.

5. Hugouvieux V, Kwak JM, Schroeder JI: An mRNA cap bindingprotein, ABH1, modulates early abscisic acid signal transductionin Arabidopsis. Cell 2001, 106:477-487.

6. Xiong L, Gong Z, Rock CD, Subramanian S, Guo Y, Xu W,Galbraith D, Zhu JK: Modulation of abscisic acid signaltransduction and biosynthesis by an Sm-like protein inArabidopsis. Dev Cell 2001, 1:771-781.

7. Staiger D: RNA-binding proteins and circadian rhythms inArabidopsis thaliana. Philos Trans R Soc Lond B Biol Sci 2001,356:1755-1759.

8. Cole CN: Choreographing mRNA biogenesis. Nat Genet 2001,29:6-7.

9. Proudfoot NJ, Furger A, Dye MJ: Integrating mRNA processing with • transcription. Cell 2002, 108:501-512.This review cogently summarizes recent findings on the mechanisms thatensure the production of complete, fully processed, error-free transcripts.

10. Reed R, Hurt E: A conserved mRNA export machinery coupled to • pre-mRNA splicing. Cell 2002, 108:523-531.This review focuses on the mechanisms that transport mRNAs from thenucleus to the cytoplasm, and on how they are integrated with the processingof transcripts.

11. Kloc M, Zearfoss NR, Etkin LD: Mechanisms of subcellular mRNA • localization. Cell 2002, 108:533-544.This review summarizes our rapidly expanding knowledge of how RNA-binding proteins are involved in determining polarity within cells, a crucialprocess that underlies differentiation.

12. Johnstone O, Lasko P: Translational regulation and RNA • localization in Drosophila oocytes and embryos. Annu Rev Genet

2001, 35:365-406.This review describes some of the best-studied Drosophila RNA-bindingproteins and the multiple ways in which they participate in determiningembryo polarity.

13. Zhong J, Peters AH, Kafer K, Braun RE: A highly conserved sequenceessential for translational repression of the protamine 1 messengerRNA in murine spermatids. Biol Reprod 2001, 64:1784-1789.

14. Brennan CM, Steitz JA: HuR and mRNA stability. Cell Mol Life Sci2001, 58:266-277.

15. Mitchell P, Tollervey D: mRNA turnover. Curr Opin Cell Biol 2001, • 13:320-325.This review provides information on the development of mature mRNPs, aswell as on mRNA surveillance systems and mRNA degradation.

16. Nishikura K: A short primer on RNAi: RNA-directed RNA • polymerase acts as a key catalyst. Cell 2001, 107:415-418.The author provides a brief overview of the emerging field of RNA degradationtriggered by dsRNA.

17. Moore MJ: Nuclear RNA turnover. Cell 2002, • 108:431-434.Much of the RNA synthesized in the cell is excised from transcripts anddegraded without ever leaving the nucleus. This review focuses on intranuclearRNA metabolism.

18. Bachand F, Triki I, Autexier C: Human telomerase RNA–proteininteractions. Nucleic Acids Res 2001, 29:3385-3393.

19. Boumil RM, Lee JT: Forty years of decoding the silence inX-chromosome inactivation. Hum Mol Genet 2001, 10:2225-2232.

20. Anantharaman V, Koonin EV, Aravind L: Comparative genomics andevolution of proteins involved in RNA metabolism. Nucleic AcidsRes 2002, 30:1427-1464.

21. Wickens M, Bernstein DS, Kimble J, Parker R: A PUF family portrait:3′′UTR regulation as a way of life. Trends Genet 2002, 18:150-157.

22. Ostareck-Lederer A, Ostareck DH, Hentze MW: Cytoplasmicregulatory functions of the KH-domain proteins hnRNPs K andE1/E2. Trends Biochem Sci 1998, 23:409-411.

23. Perez-Canadillas JM, Varani G: Recent advances in RNA–proteinrecognition. Curr Opin Struct Biol 2001, 11:53-58.

24. Geiss G, Jin G, Guo J, Bumgarner R, Katze MG, Sen GC:A comprehensive view of regulation of gene expression bydouble-stranded RNA-mediated cell signaling. J Biol Chem 2001,276:30178-30182.

25. Ferrandon D, Koch I, Westhof E, Nusslein-Volhard C: RNA–RNAinteraction is required for the formation of specific bicoid mRNA3′′UTR-STAUFEN ribonucleoprotein particles. EMBO J 1997,16:1751-1758.

26. Ramos A, Grunert S, Adams J, Micklem DR, Proctor MR, Freund S,Bycroft M, St Johnston D, Varani G: RNA recognition by a Staufendouble-stranded RNA-binding domain. EMBO J 2000, 19:997-1009.

27. Perrone-Bizzozero N, Bolognani F: Role of HuD and otherRNA-binding proteins in neural development and plasticity.J Neurosci Res 2002, 68:121-126.

28. Wilkinson MF, Shyu AB: Multifunctional regulatory proteins that •• control gene expression in both the nucleus and the cytoplasm.

Bioessays 2001, 23:775-787.This is an interesting overview that focuses on the ability of certain kinds ofproteins to influence and integrate different aspects of regulation. Theauthors point out that some of the proteins originally identified as transcriptionfactors have important regulatory functions at the RNA level.

29. St Johnston D, Beuchle D, Nusslein-Volhard C: Staufen, a generequired to localize maternal RNAs in the Drosophila egg. Cell1991, 66:51-63.

30. St Johnston D, Nusslein-Volhard C: The origin of pattern andpolarity in the Drosophila embryo. Cell 1992, 68:201-219.

31. Brendza RP, Serbus LR, Duffy JB, Saxton WM: A function forkinesin I in the posterior transport of oskar mRNA and Staufenprotein. Science 2000, 289:2120-2122.

32. Schnorrer F, Bohmann K, Nusslein-Volhard C: The molecular motor •• dynein is involved in targeting swallow and bicoid RNA to the

anterior pole of Drosophila oocytes. Nat Cell Biol 2000, 2:185-190.An interesting and important analysis of the mechanism by which proteinsare localized in cells as a result of mRNA localization mediated by theinteraction of RNA-binding proteins with motor proteins.

33. Shen CP, Knoblich JA, Chan YM, Jiang MM, Jan LY, Jan YN: Mirandaas a multidomain adapter linking apically localized Inscuteableand basally localized Staufen and Prospero during asymmetriccell division in Drosophila. Genes Dev 1998, 12:1837-1846.

34. Gunkel N, Yano T, Markussen FH, Olsen LC, Ephrussi A:Localization-dependent translation requires a functionalinteraction between the 5′′ and 3′′ ends of oskar mRNA. GenesDev 1998, 12:1652-1664.

35. Krichevsky AM, Kosik KS: Neuronal RNA granules: a link betweenRNA localization and stimulation-dependent translation. Neuron2001, 32:683-696.

36. Micklem DR, Adams J, Grunert S, St Johnston D: Distinct roles of •• two conserved Staufen domains in oskar mRNA localization and

translation. EMBO J 2000, 19:1366-1377.This study dissects the Staufen protein, illustrating how different parts ofthe protein are required for localizing the RNA in the embryo and regulatingits translation.

37. Wagner C, Palacios I, Jaeger L, St Johnston D, Ehresmann B,Ehresmann C, Brunel C: Dimerization of the 3′′UTR of bicoid mRNAinvolves a two-step mechanism. J Mol Biol 2001, 313:511-524.

38. Lorkovic ZJ, Barta A: Genome analysis: RNA recognition motif(RRM) and K homology (KH) domain RNA-binding proteins fromthe flowering plant Arabidopsis thaliana. Nucleic Acids Res 2002,30:623-635.

39. Jensen KH, Herrin DL, Plumley FG, Schmidt GW: Biogenesis ofphotosystem II complexes: transcriptional, translational, andposttranslational regulation. J Cell Biol 1986, 103:1315-1325.

40. Rochaix JD, Kuchka M, Mayfield S, Schirmer-Rahire M, Girard-Bascou J,Bennoun P: Nuclear and chloroplast mutations affect thesynthesis or stability of the chloroplast psbC gene product inChlamydomonas reinhardtii. EMBO J 1989, 8:1013-1021.

41. Danon A, Mayfield SP: Light regulated translational activators:identification of chloroplast gene specific mRNA binding proteins.EMBO J 1991, 10:3993-4001.

42. Yohn CB, Cohen A, Danon A, Mayfield SP: A poly(A) binding proteinfunctions in the chloroplast as a message-specific translationfactor. Proc Natl Acad Sci USA 1998, 95:2238-2243.

43. Mayfield SP, Cohen A, Danon A, Yohn CB: Translation of the psbAmRNA of Chlamydomonas reinhardtii requires a structured RNAelement contained within the 5′′ untranslated region. J Cell Biol1994, 127:1537-1545.

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44. Yohn CB, Cohen A, Danon A, Mayfield SP: Altered mRNA bindingactivity and decreased translational initiation in a nuclear mutantlacking translation of the chloroplast psbA mRNA. Mol Cell Biol1996, 16:3560-3566.

45. Zerges W, Rochaix JD: Low density membranes are associatedwith RNA-binding proteins and thylakoids in the chloroplast ofChlamydomonas reinhardtii. J Cell Biol 1998, 140:101-110.

46. Tarun SZ Jr, Sachs AB: Association of the yeast poly(A) tail bindingprotein with translation initiation factor eIF-4G. EMBO J 1996,15:7168-7177.

47. Trebitsh T, Levitan A, Sofer A, Danon A: Translation of chloroplast •• psbA mRNA is modulated in the light by counteracting oxidizing

and reducing activities. Mol Cell Biol 2000, 20:1116-1123.This work shows how the oxidation and reduction of an RNA-binding proteinregulates the translation of psbA RNA.

48. Danon A, Mayfield SP: ADP-dependent phosphorylation regulatesRNA-binding in vitro: implications in light-modulated translation.EMBO J 1994, 13:2227-2235.

49. Stitt M, Wirtz W, Heldt HW: Metabolite levels during induction inthe chloroplast and extrachloroplast compartments of spinachprotoplasts. Biochim Biophys Acta 1980, 593:85-102.

50. Danon A, Mayfield SP: Light-regulated translation of chloroplastmessenger RNAs through redox potential. Science 1994,266:1717-1719.

51. Trebitsh T, Danon A: Translation of chloroplast psbA mRNA isregulated by signals initiated by both photosystems II and I.Proc Natl Acad Sci USA 2001, 98:12289-12294.

52. Carlberg I, Rintamaki E, Aro EM, Andersson B: Thylakoid proteinphosphorylation and the thiol redox state. Biochemistry 1999,38:3197-3204.

53. Shen Y, Danon A, Christopher DA: RNA binding-proteins interactspecifically with the Arabidopsis chloroplast psbA mRNA5′′ untranslated region in a redox-dependent manner. Plant CellPhysiol 2001, 42:1071-1078.

54. Salvador ML, Klein U: The redox state regulates RNA degradationin the chloroplast of Chlamydomonas reinhardtii. Plant Physiol1999, 121:1367-1374.

55. Nickelsen J, van Dillewijn J, Rahire M, Rochaix JD: Determinants forstability of the chloroplast psbD RNA are located within its shortleader region in Chlamydomonas reinhardtii. EMBO J 1994,13:3182-3191.

56. Boudreau E, Nickelsen J, Lemaire SD, Ossenbuhl F, Rochaix JD:The Nac2 gene of Chlamydomonas encodes a chloroplastTPR-like protein involved in psbD mRNA stability. EMBO J 2000,19:3366-3376.

57. Vaistij FE, Boudreau E, Lemaire SD, Goldschmidt-Clermont M,Rochaix JD: Characterization of Mbb1, a nucleus-encodedtetratricopeptide-like repeat protein required for expression of thechloroplast psbB/psbT/psbH gene cluster in Chlamydomonasreinhardtii. Proc Natl Acad Sci USA 2000, 97:14813-14818.

58. Petracek ME, Dickey LF, Nguyen TT, Gatz C, Sowinski DA, Allen GC,Thompson WF: Ferredoxin-1 mRNA is destabilized by changes inphotosynthetic electron transport. Proc Natl Acad Sci USA 1998,95:9009-9013.

59. Abler ML, Green PJ: Control of mRNA stability in higher plants.Plant Mol Biol 1996, 32:63-78.

60. Newman TC, Ohme-Takagi M, Taylor CB, Green PJ: DST sequences,highly conserved among plant SAUR genes, target reportertranscripts for rapid decay in tobacco. Plant Cell 1993, 5:701-714.

61. Johnson MA, Perez-Amador MA, Lidder P, Green PJ: Mutants ofArabidopsis defective in a sequence-specific mRNA degradationpathway. Proc Natl Acad Sci USA 2000, 97:13991-13996.

62. Lewis JD, Izaurralde E: The role of the cap structure in RNAprocessing and nuclear export. Eur J Biochem 1997, 247:461-469.

63. Kmieciak M, Simpson CG, Lewandowska D, Brown JW, Jarmolowski A:Cloning and characterization of two subunits of Arabidopsisthaliana nuclear cap-binding complex. Gene 2002, 283:171-183.

64. Dostie J, Ferraiuolo M, Pause A, Adam SA, Sonenberg N: A novelshuttling protein, 4E-T, mediates the nuclear import of the mRNA5′′ cap-binding protein, eIF4E. EMBO J 2000, 19:3142-3156.

65. McKendrick L, Thompson E, Ferreira J, Morley SJ, Lewis JD:Interaction of eukaryotic translation initiation factor 4G with thenuclear cap-binding complex provides a link between nuclear andcytoplasmic functions of the m(7) guanosine cap. Mol Cell Biol2001, 21:3632-3641.

66. Sachs AB: Cell cycle-dependent translation initiation: IRESelements prevail. Cell 2000, 101:243-245.

67. Sheen J: Mutational analysis of protein phosphatase 2C involvedin abscisic acid signal transduction in higher plants. Proc NatlAcad Sci USA 1998, 95:975-980.

68. Bouveret E, Rigaut G, Shevchenko A, Wilm M, Seraphin B: A Sm-like • protein complex that participates in mRNA degradation. EMBO J

2000, 19:1661-1671.This study is an elegant demonstration of how to resolve two multi-proteincomplexes of overlapping protein composition but different functions.

69. Tena G, Asai T, Chiu W-L, Sheen J: Plant mitogen-activated protein kinase signaling cascades. Curr Opin Plant Biol 2001,4:392-400.

70. Lopez-Molina L, Chua NH: A null mutation in a bZIP factor confersABA-insensitivity in Arabidopsis thaliana. Plant Cell Physiol 2000,41:541-547.

71. Lopez-Molina L, Mongrand S, Chua NH: A post-germination •• developmental arrest checkpoint is mediated by abscisic acid and

requires the ABI5 transcription factor in Arabidopsis. Proc NatlAcad Sci USA 2001, 98:4782-4787.

This nice study begins to reveal the molecular mechanism underlying theability of ABA to arrest development just after germination. This is the propertythat is most commonly used to identify mutations in ABA signaling.

72. Kang J-Y, Choi H-I, Im M-Y, Kim SY: Arabidopsis basic leucinezipper proteins that mediate stress-responsive abscisic acidsignaling. Plant Cell 2002, 14:343-357.

73. Laroia G, Cuesta R, Brewer G, Schneider RJ: Control of mRNAdecay by heat shock-ubiquitin-proteasome pathway. Science1999, 284:499-502.

74. Cuesta R, Laroia G, Schneider RJ: Chaperone hsp27 inhibitstranslation during heat shock by binding eIF4G and facilitatingdissociation of cap-initiation complexes. Genes Dev 2000,14:1460-1470.

75. Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M: Auxin regulatesSCFTIR1-dependent degradation of AUX/IAA proteins. Nature2001, 414:271-276.