THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 15, Ieaue of August 10, pp. 9929-9935,1984 Prinkd in U.S.A.

Apolipoprotein I1 Messenger RNA TRANSCRIPTIONAL AND SPLICING HETEROGENEITY YIELDS SIX 5”UNTRANSLATED LEADER SEQUENCES*

(Received for publication, March 9, 1984)

Gregory S. ShelnessS and David L. Williams4 From the Department of Pharmacological Sciences, State University of New York ut Stony Brook, Stony Brook, New York 11 794-8651

The structure of the mRNA for apolipoprotein I1 (apo-11), a major avian estrogen-responsive yolk pro- tein, has been investigated. Primer extension using a cDNA probe-primer revealed six forms of mature apo- I1 mRNA. S1 and primer extension analysis with in- tron probes showed that only three of these forms arise due to multiple sites of transcription initiation on the apo-I1 gene. To investigate the basis for the additional forms of apo-I1 mRNA, we examined their nucleotide sequence. The sequence obtained between -10 and -42, relative to translation initiation, is identical to that reported by Wieringa et d. (Wieringa, B., AB, G., and Gruber, M. (1981) Nucleic AcidsRes. 9,469-499). At position -43, however, sequence heterogeneity ap- pears. The minor form of the sequence starting at -43 and extending in a 5’ direction corresponds exactly to the published mRNA sequence. The major form of the sequence has the insertion, 5’-CAG-3’, at this position. The apparent basis for this heterogeneity is an unusual intron-exon border which violates the consensus se- quence found in comparable positions in most eukar- yotic genes by the existence of two adjacent 5’-CAG- 3‘ triplets after the pyrimidine (Y)-rich track. The processing ratio between intron removal at the up- stream and downstream AG dinucleotide is approxi- mately 2.5:l. This result demonstrates that the splicing mechanism allows spatial flexibility in the positioning of the highly conserved YAG triplet within the consen- sus sequence at the 3’ splice site.

~ ~~ ~

In the laying hen, apo-11’ comprises approximately 50% of the protein of plasma very low density lipoprotein (1). In the rooster and the immature hen, plasma apo-I1 levels are neg- ligible (1-10 ng/ml) (2) but increase by 106-107-fold in re- sponse to exogenous estrogen (1-3). Accompanying this mas- sive accumulation of apo-11 in the plasma is a dramatic increase in both hepatic apo-I1 synthesis and the concentra- tion of apo-I1 mRNA (1). As is the case with other oviparate estrogen-regulated reproductive proteins (4-9), the effects of estrogen on hepatic apo-I1 mRNA levels have been attributed

* This research was supported by National Institutes of Health Grant AM18171. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Predoctoral Traineeship GM07518 in Pharmacological Sciences. To whom correspondence should be directed. The abbreviations used are: apo-11, apolipoprotein II; bp, base

pair; kbp, kilobase pair; ddNTP, dideoxynucleotide triphosphate; AMV, avian myeloblastosis virus; Y, any pyrimidine; 3’-SS, 3‘ splice site.

to transcriptional activation of the gene as well as a selective stabilization of its mRNA in the cytoplasm (10).

Apo-I1 is well suited for the analysis of estrogen-regulated gene expression due to the compact size of the apo-I1 gene (11,12) and its mRNA (13) (approximately 3000 bp and 600 bases, respectively) as well as its dramatic response to hor- mone. Work to date demonstrates that apo-I1 mRNA tran- scription is initiated at multiple sites on the gene (14, 15) although there is disagreement with respect to the actual number of these sites and their orientation relative to the gene sequence (14, 15).

In the present report, we show that mature apo-I1 mRNA consists of at least six species with different 5”untranslated leader sequences. Comparison of the mature mRNAs and their nuclear precursors shows that the six leader sequences result from three sites of transcription initiation on the apo-I1 gene and two pathways for splicing of the precursor mRNA at the first intron-exon junction. Alternate splicing at this site may reflect the precision of the splicing mechanism when pre- sented with adjacent CAG triplets (5’ . . . CAGCAG . . . 3’) at an intron-exon border (16, 17).

EXPERIMENTAL PROCEDURES

Materials-Restriction enzymes, T4 DNA ligase, T4 polynucleo- tide kinase, bacterial alkaline phosphatase, and nuclease S1 were from Bethesda Research Laboratories, Inc., Gaithersburg, MD or New England Biolabs, Beverly, MA. ddNTPs and dNTPs were from P-L Biochemicals, Inc., Milwaukee, WI. AMV reverse transcriptase was obtained from Life Sciences, Inc., St. Petersburg, FL. Except where noted, all other chemicals were purchased from Sigma Bio- chemical, Inc., St. Louis, MO or Fisher scientific, Fair Lawn, NJ.

Animals-White leghorn roosters (SPAFAS, Norwich, CT), re- ceived an intramuscular injection of 17P-estradiol (25 mg/kg of body weight) in propylene glycol approximately 72 h before being killed

Apo-ZI cDNA Clone-The isolation and characterization of an apo- I1 cDNA clone have been described (18). This clone is designated apo-1153.8.

Isolation of a Genomic Apo-II Ctone-A genomic chicken DNA library (19) consisting of recombinant X Charon 4A phages was kindly supplied by R. Axel (Columbia University, New York). The library was screened by an in situ plaque hybridization technique (20) using a 32P-labeled 240-bp PstI fragment derived from clone apo-I1 53.8. After plaque purification and DNA isolation, the apo-I1 genomic clone, X apo-I1 A, was characterized by restriction analysis. The restriction map obtained for X apo-I1 A is identical to that obtained by Wiskocil et al. (11) for their genomic apo-I1 clone, apo-I1 b.

Subcloning of DNA Restrietwn Fragmnts-DNA from recombi- nant clone X apo-I1 A was digested with EcoRI. Following ligation to EcoRI-digested and dephosphorylated pBR325 (21), the recombinant plasmids were used to transform Escherichia coli K12 strain HBlOl by the method of Dagert and Ehrlich (22). A plasmid containing a 5.6-kbp DNA insert (p5apoIIAR-5.6) was identified by restriction analysis and its hybridization with the appropriate restriction frag- ment from X apo-I1 A. This insert contains several kilobase pairs of 5’-flanking sequence, the entire leader exon (exon I), and approxi-

9929

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9930 Transcription and Processing of Apo-II mRNA mately 0.75 kbp of the first intron (intron A) (see Fig. 3 and Refs. 12, 13, and 15 for organization and sequence of the apo-I1 gene).

An M13 subclone containing the noncoding strand of the apo-I1 gene sequence from -484 - +256 was constructed as follows. Sub- clone p5apoIIAR-5.6 was digested with PstI and BglII and the prod- ucts were separated on a 1% agarose gel (23). The 740-bp fragment was electroeluted (24), ligated to BamHI-Pstl-digested M13mp8 (rep- licative form) (25), and used to transform E. coli K12 strain JM103. The identity of the M13 subclone, mpSapoIIABP, was verified by sequence analysis.

Kinase Labeling of Probes-Probe A (Fig. 1) was prepared by digestion of apo-I1 53.8 (18) with HinfI followed by electrophoresis through a 0.7% agarose gel. The 1.4-kbp fragment was recovered by electroelution, dephosphorylated with bacterial alkaline phosphatase, and labeled at the 5' end with [y-32P]ATP (3000 Ci/mmol, Amersham Corp., Arlington Heights, IL) and polynucleotide kinase (26). This fragment contains 46 nucleotides from the 5' end of the apo-I1 cDNA insert, 23 nucleotides of poly(G) linker, as well as approximately 1.35 kbp of pBR322. Following secondary digestion with Had11 (the HaeIII restriction site is formed artifically at the junction of the poly(G) linker used to insert the double-stranded cDNA into the Pst- I site of pBR322 and the C-C dinucleotide in the mRNA sequence at -9,-10) (13,18), the fragments were electrophoresed in a denaturing 6% polyacrylamide gel (26). The 46-base HaeIII-HinfI fragment was visualized by autoradiography and eluted according to the method of Maxam and Gilbert (26).

Genomic probes B and C (Fig. 3) were prepared by digestion of subclone p5apoIIAR-5.6 with BstEII followed by dephosphorylation and 5' end-labeling as described above. Probe B was generated by secondary digestion with AvaII whereas probe C was produced by HinfI digestion. Gel purification of probes B and C was as described above.

Primer Extension Using cDNA Probe-PrimerA-5,000-10,000 cpm (Cerenkov) of 5' end-labeled probe primer was combined with 3 pg of estrogen-induced total liver RNA in 10 pl of buffer A (50 mM Tris- HC1, pH 8.3 at 42 "C, 540 mM KCl). Samples were incubated at 60 "C for 1 h. After cooling to room temperature, each sample was adjusted such that the final buffer conditions were 50 mM Tris-HC1, pH 8.3 at 42 "C, 135 mM KCI, 10 mM MgCl,, 0.7 mM dGTP, dATP, dTTP, and dCTP, and 5 mM dithiothreitol (final volume, 40 pl). After addition of 8 units of AMV reverse transcriptase, samples were incubated at 42 "C for 1.5 h. Reactions were quenched by the addition of 260 pl of 300 mM Na acetate, 10 mM Tris-HC1, pH 7.5, 1 mM EDTA, and 600 pl of cold 100% ethanol. Following incubation at -70 'C for 30 min, the DNA was pelleted in an Eppendorf microcentrifuge (5 min, 4 'C).

Primer Extension Using Genomic Probe-Primer B-30,000 cpm (Cerenkov) of 5' end-labeled probe-primer was combined with 250 pg of total liver RNA in 200 pl of buffer A containing 1 mM EDTA. Samples were incubated 16 h at 60 "C. After diluting with 1 volume of H20, the samples were precipitated with 2 volumes of 100% ethanol, washed with 100% ethanol, and dried. Pellets were dissolved in water and brought to the same AMV reverse transcriptase assay conditions described above (final volume, 250 pl). 30 units of AMV reverse transcriptase were added to each sample and incubation was carried out for 1.5 h at 42 "C. Following the addition of EDTA to 15 mM, the reaction was made 0.2 N NaOH and incubation was continued for 1 h. After neutralization with HCl, samples were phenol extracted and ethanol precipitated. SI Nuclease Mapping of Precursor mRNA"S1 nuclease mapping

was performed essentially by the method of Berk and Sharp (27) as modified by Weaver and Weissmann (28). 100,000 cpm (Cerenkov) of 32P end-labeled probe C were combined with 250 pg of liver RNA in 200 pl of 80% deionized formamide, 400 mM NaC1,40 mM pipera- zine-N,N'-bis(2-ethanesulfonic acid), pH 6.4, and 1 mM EDTA. Samples were incubated for 16 h a t 56 "C. After 10-fold dilution into S1 nuclease buffer (400 mM NaCl, 30 mM sodium acetate, pH 4.8, 1 mM ZnClz, 10 pg/ml denatured salmon sperm DNA), nuclease S1 was added to a final concentration of 500-3000 units/ml and samples were incubated for 1 h at 30 "C. After acijusting the EDTA concen- tration to 15 mM and the pH to 7, the nucleic acid was ethanol precipitated. The S1-protected fragments were further purified oy treatment with 0.2 N NaOH, followed by neutralization, phenol extraction, and ethanol precipitation as described above for primer extension mapping of apo-I1 precursor mRNC,.

Denaturing Gel Electrophoresis-Washed (95% ethanol) and dried DNA pellets were dissolved in gel-loading solution (95% deionized formamide, 20 mM EDTA, and dyes), boiled for 2 min and placed on

ice. Samples were analyzed by electrophoresis through 0.4-mm-thick denaturing polyacrylamide gels as described by Maxam and Gilbert (26). For autoradiography, gels were dried and exposed to Kodak XAR-5 film (-70 "C) using a DuPont Cronex intensifying screen.

RNA Sequencing-cDNA-primed dideoxy-RNA sequencing (29) was performed using the same conditions described above for primer extension but with the following ddNTP additions: G reaction, 5 p~ ddGTP; A reaction, 5 p~ ddATP; T reaction, 5 p~ ddTTP C reaction, 2.5 p~ ddCTP. The corresponding dNTP concentration in each reaction was 25 p ~ . The remaining dNTPs were included at 100 p ~ . Following a 25-min incubation at 42 "C, the concentration of each dNTP was raised to 700 pM and incubation was continued for another 15 min.

DNA Sequencing-Sequencing of single-stranded M13 DNA was by the method of Sanger et al. (30). Pentadecamer single-stranded M13 sequencing primer was from New England Biolabs.

Liquid Scintillation Counting of Gel Slices-Gel slices were excised from denaturing polyacrylamide gels and incubated in 0.5 ml of 30% hydrogen peroxide for 48 h at 50 "C. Samples were counted following addition of 7.5 ml of Liquiscint (National Diagnostics, Somerville, NJ).

Trichloroacetic Acid Precipitation of Probe DNA-Since large amounts of liver RNA are used for the precursor-mapping experi- ments, samples are contaminated with polysaccharide which pre- cludes adequate concentration of the sample prior to gel electropho- resis. To purify probe DNA from polysaccharide, we have used the following procedure. After final ethanol precipitation, the DNA pel- lets are dissolved in 250 pl of H20. An equal volume of ice-cold 2.5% trichloroacetic acid is added, and precipitation is allowed to proceed for 10 min on ice. After centrifugation for 5 min (Eppendorf micro- centrifuge, 4 "C) the pellets are washed twice with 1.5 ml of 70% ethanol, 150 mM NaCl and once with 100% ethanol. Samples are dried and dissolved in gel-loading solution. Control experiments revealed that the electrophoretic mobilities of DNA fragments are unchanged following acid precipitation (data not shown).

RESULTS

Mapping the 5' Terminus of Apo-11 mRNA by Primer Extension-To date, two laboratories have used S1 nuclease mapping to characterize mRNA precursor molecules tran- scribed from the apo-I1 gene. van het Schip et al. (15) observed two primary transcripts whose 5' ends map to -11 and +1. Hache et al. (14) detected three transcripts originating at -19, -9, and +8 (with respect to the +1 initiation site mapped by van het Schip et aL). Our results will be described only in terms of the transcription coordmates of van het Schip et al. (15).

Total cellular RNA used in the experiments described below was purified from estrogen-treated rooster liver as described (18). To precisely map the 5' end of the mature apo-I1 message, primer extension (31-33) was performed following the experimental strategy shown in Fig. 1. The 5' end-labeled cDNA primer used in this experiment (probe A) is derived

m'Gppp 4 (A), apon rnRNA -. probe A

(G)23 ' ' -10 +36

H Hf ( Q Z 3 o p o n 53.8

FIG. 1. Strategy for mapping the 5' terminus of apo-I1 mRNA by primer extension. Probe A and the apo-I1 cDNA clone (apoZZ 53.8) from which it is derived are shown aligned with the apo- I1 mRNA (13). The shuded area indicates the protein-coding region and the triangular box delineates the splice junction formed by the joining of RNA encoded by the first two exons of the gene (see Fig. 3 and Refs. 11, 12, and 15 for organization and sequence of the apo-I1 gene). The single stranded 5' end-labeled probe A (* indicates location of 32P label) is synthesized and annealed to 3 pg of liver RNA as described under "Experimental Procedures." Following treatment with reverse transcriptase in the presence of the four deoxynucleotide triphosphates, the extended products are sized by denaturing gel electrophoresis. The size of the extended probe should correspond to the distance between position +36 on the mRNA and its 5' terminus.

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Transcription and Processing of Apo-11 mRNA 9931

from apo-I1 clone 53.8. As indicated, the 3‘ end of probe A maps on the apo-I1 mRNA to position -10 with respect to the initiation of translation (13) and is situated approximately 30 nucleotides 3‘ to the reported splice junction formed by the joining of RNA encoded by exons I and I1 (Fig. 1) (15). Analysis of the extended cDNA products by denaturing 10% polyacrylamide gel electrophoresis followed by autoradiogra- phy demonstrates a cluster of six doublets, each consisting of two cDNA products separated by a single nucleotide (Fig. 2, lanes 1 and 2). The production of artifactual doublets during primer extension has been reported previously (32-34) and is thought to result from premature termination by reverse transcriptase as it encounters a 5’ cap structure (35). If each doublet represents a single 5’ terminus, this result suggests the existence of six species of mature apo-I1 mRNA designated in order of increasing mobility of their corresponding cDNAs as IA, IB, 2A, 2B, 3A, and 3B (Fig. 2). The basis for this nomenclature will become apparent below.

In order to accurately size the six extended products, we compared their gel mobilities with a dideoxynucleotide-se- quencing ladder (30) generated from an M13 clone which contains an apo-I1 cDNA sequence from another region of the apo-I1 mRNA. (Fig. 2, lanes G, A, T, and C ) . The use of such a “nonhomologous” single nucleotide marker system was nec- essary since our cDNA clone as well as the other apo-I1 cDNA clones reported in the literature (10, 13, 35) do not extend to the 5’ end of the mRNA. The use of this marker system allows us to size DNA fragments within the size range of interest to within k0.5 nucleotides as determined by control experiments using apo-I1 cDNA (13) and pBR322 (36) restric- tion fragments of known length. With this procedure, the length in bases of the upper band of each cDNA doublet was determined and is shown in Fig. 2. Utilizing knowledge of the reported splice junctions (15), and assuming no 5’ processing of the primary transcripts (37), the 3’ ends of the primer extended products map to the indicated positions on the apo I1 gene. As shown in Fig. 2, only two of the six products map to positions reported as transcription initiation sites identified by S1 nuclease mapping with intron-specific hybridization probes (14, 15). This result suggests that the mature apo-I1 mRNA is considerably more complex at the 5’ end than the primary transcription products. Note also, as determined by additional results reported below, that the heterogeneity of the primer-extended products does not reflect transcriptional pausing by reverse transcriptase on the RNA template.

SI and Primer Extension Mapping of the Apo-II mRNA Precursor-To further investigate the basis of the observed apo-I1 mRNA heterogeneity, we mapped the 5’ ends of the apo-I1 mRNA precursors by S1 and primer extension mapping using intron-specific hybridization probes. The use of primer extension and S1 probes which share a common 5’ end should, after the respective mapping procedures, yield DNAs of iden- tical electrophoretic mobility. The two probes used in this experiment were constructed from genomic subclone p5apoIIAR-5.6 (Fig. 3 and “Experimental Procedures”) and were 5’ end labeled a t a common BstEII site situated within the first intron (intron A). The primer extension probe (probe B) extends in a 5‘ direction from the labeled BstEII site to an AuaII restriction site still within the first intron. The S1 probe (probe C) extends to a Hinfl site located well upstream from the region of transcription initiation (Fig. 3).

Probes B and C were independently hybridized to 250 pg of total liver RNA as described under “Experimental Proce- dures.” After treatment of the probe-RNA hybrids with re- verse transcriptase or nuclease S1, the resultant DNA prod- ucts were purified and analyzed by electrophoresis in a 6%

A B

1A- 16- 2A - 26- 3A- 30-

G A l T C 2 G A l T C 2

-127- 0 -1 24-

t-116--, -113- -110- -107-

P r r -

5’ A T G C C A 0 G T G

T C A I

A 0 T G

G A

A G O C T T C A G C C 3’

FIG. 2. Mapping the 5’ terminus of apo-I1 mRNA by primer extension. Probe A was hybridized to total liver RNA and extended with reverse transcriptase as described under “Experimental Proce- dures.” The extended products were analyzed by denaturing 10% polyacrylamide gel electrophoresis followed by autoradiography for 24 (A ) or 72 h (B) . Lanes 1 and 2 show independent primer extensions on the same sample of RNA. Lanes G, A, T, and C are the individual dideoxynucleotide-sequencing reactions used to generate a nonho- mologous single nucleotide marker system as described in the text. Numbers listed between A and B and aligned with each extended product in lanes 1 and 2 indicate the length of the extended products in bases (k0.5). The two character labels to the left of A and also aligned with the extended products are the designations used to identify each form of the mRNA template which gives rise to the corresponding extended cDNA product. The sequence shown on the right is the 5’ region of the apo-I1 gene (14, 15). Lines are drawn between the apparent transcription initiation sites on the gene and the corresponding extended product using assumptions explained in the text (lines are drawn between adjacent nucleotides in the sequence to indicate the k0.5 nucleotide error in sizing the fragments). 0 next to the sequence indicates transcription initiation sites mapped by Hache et al. (14). A indicates transcription initiation sites mapped by van het Schip et al. (15). None of the extended products identified in this analysis arise when no RNA or control liver RNA is used as template (data not shown). Under the electrophoresis conditions used, the 46-base probe-primer A is run off the gel.

denaturing polyacrylamide gel. As shown in Fig. 4, lanes 1-3, both mapping procedures strongly suggest the existence of three forms of the apo-I1 mRNA precursor as evidenced by three groups of prominent bands indicated with numbered arrows. Examination of short (Fig. 4A) and long (Fig. 4B)

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9932 Transcription and Processing of Apo-II mRNA

- I kb

Hinf I c

Est 1E II I - 1

- probe B probe C

100 bp

FIG. 3. Strategy for mapping the 5’ ends of apo-I1 precursor mRNAs with primer extension probe B and S1 nuclease probe C. The apo-I1 genomic clone, X apo-I1 A, is shown at the top. The intron-exon organization (11, 12, 15) is indicated by shuded boxes (exons) and unbroken lines (introns and intergenic regions). The broken lines indicate the vector arms. An expanded diagram of the region around the small leader exon (exon I) is shown just below (note difference in scale). Genomic subclone p5apoIIAR-5.6 contains the 5.6 kbp of genomic DNA between EcoRI (R l ) sites 2 and 3. This subclone is used to generate the 5’ end-labeled probes B and C (* indicates the position of the 3zP label) as described under “Experi- mental Procedures.”

autoradiographic exposures of the gel reveals that each num- bered set of SI-protected products (lanes 1 and 2 ) is composed primarily of two bands separated by a single nucleotide. In contrast, the primer extension mapping (lane 3) demonstrates three discrete products whose mobilities are identical to the lower band of each S1 doublet. This result suggests that S1 has difficulty removing the final single-stranded nucleotide a t the junction between the single- and double-stranded region of the RNA-probe C hybrid. Such artifactual protection of a single-stranded S1 probe has been reported previously (28, 38). Note also that prolonged exposure of the gel (Fig. 4B) shows minor bands in the S1 lanes which are not produced by primer extension. These bands, therefore, are probably artifacts unique to the S1 protection assay.

The three bands ( 1 , 2, and 3) common to both the primer extension and S1 analysis were oriented with respect to the apo-I1 gene by comparison with a sequencing ladder (Fig. 4, lanes G, A , T, and C) generated by annealing probe B to M13 subclone mpSapoIIABP, which contains the noncoding strand of the appropriate region of the apo-I1 gene, followed by dideoxy sequencing (38). As shown in Fig. 4, the 5‘ ends of the mRNA precursors map to positions -11, +1, and +7 with respect to the major cap site determined by van het Schip et al. (15). The relative abundance of each precursor based on direct counting of each form of S1 and reverse transcriptase- modified probe is 15% (-11), 80% (+l), and 5% (+7). This result suggests that only three of the six species of mature apo-I1 message identified in Fig. 2 can be explained by mul- tiple sites of transcription initiation. The biosynthesis of three of the mature mRNAs is consistent with our precursor map- ping studies (Fig. 4) as well as the reported splice sites (15) used to remove intron A. These are identified in Fig. 2 as 1B, 2B, and 3B. Each of the three remaining apo-I1 mRNAs, IA, 2A, and 3A, is three nucleotides longer than forms IB, 2B, and 3B, respectively. This suggests that forms l A , 2A, and 3A may arise, via an alternate splicing pathway, from the same precursors used to generate 1 B, 2B, and 3B.

Apo-II mRNA Splicing Heterogeneity-To investigate the possibility that alternate splicing is responsible for the for- mation of six processed mRNAs from three precursors, we examined the nucleotide sequence of the mature apo-I1 mRNA

1 2

o m

A B

[ -11

\

5’

A

A G C A

C T

2+A +1 A C C 1

3+A +7 C

C

G T

G 3‘

FIG. 4. Parallel nuclease S1 and primer extension mapping of the 5‘ ends of apo-I1 mRNA precursors. Probes B and C, both labeled with 32P at their common BstEII restriction site, were inde- pendently hybridized to 250 pg of total liver RNA. The probe B-RNA hybrids were treated with reverse transcriptase and the probe C-RNA hybrids were treated with different concentrations of nuclease SI. Short ( A ) and long ( B ) autoradiographic exposures of the same gel are shown. Lanes 1 and 2, S1 treatment of probe C-RNA hybrids with 1500 (lane I ) , or 3000 units/ml (lane 2) of nuclease S1. Lane 3, AMV reverse transcriptase treatment of probe B-RNA hybrid. Lanes C, A, T, and C represent the sequence of the noncoding strand of the gene generated by annealing probe B to M13 genomic subclone mp9apoIIABP followed by dideoxy sequencing (38). The resultant template sequence deduced from the gel is listed along the right. Arrows 1-3 show the positions of the three forms of S1-modified probe C (lanes 1 and 2) and the reverse transcriptase-modified probe B (lane 3). Note that these products are not produced by either mapping procedure with total liver RNA from animals which have not been treated with estrogen (data not shown). The orientation of these three probe forms with respect to the apo-I1 gene is indicated by arrows to the left of the gene sequence. Note that the 5’ ends of precursors I , 2, and 3 map to the same position on the gene as the 5’ ends of mature apo-I1 forms lB, 28, and 3B, respectively (see Fig. 2 and “Results”).

population in the region of the reported splice junction formed by the joining of RNA encoded by exons I and 11. For this analysis, probe A (Fig. 1) was used as a primer for dideoxy RNA sequencing (29). The labeled products were analyzed by electrophoresis in a 6% denaturing polyacrylamide gel, fol- lowed by autoradiography. As shown in Fig. 5, a low frequency of prematurely terminated cDNA products is formed even in the absence of dideoxynucleotide triphosphates (lane -). By reading the sequence with reference to this control (-) lane it is apparent that from position -30 to position -42 the sequence obtained is identical to that published by Wieringa et al. (13). We have sequenced other regions of the mRNA by this method and also find excellent agreement with the pub- lished mRNA sequence (data not shown). Above position -42, however, sequence heterogeneity appears as evidenced by two bands in some of the ladder positions (again, the sequence must be read with reference to the control (-) lane). Since the two bands in each ladder position are always of a light and heavy intensity, the overlapping sequences can be read independently. The sequence based on careful reading of the

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Transcription and Processing of Apo-11 mRNA 9933

1 A - b A G

5'

L

" "

G ? -30 G T

FIG. 5. Sequencing the 5' end of apo-I1 mRNA. Primer exten- sion using probe A was repeated as described in Fig. 1 except that each primer extension reaction contained either no dideoxy addition (-) or was modified to include a low concentration of a dideoxynu- cleotide triphosphate (lunes C, T, A, and C). Reaction products were analyzed by denaturing 6% polyacrylamide gel electrophoresis. To the right of the autoradiogram are listed the two overlapping mRNA template sequences (nucleotides in parentheses correspond to minor processing pathway) deduced from the sequence analysis (thymine is used in place of uridine so that direct comparison can be made with the gene sequence shown in Figs. 2 and 4). The arrows next to the sequences labeled A and B show the two different junctions formed in the mature message between RNA encoded by exons I and 11. The analogous positions in the sequencing ladder are also indicated along with the positions of the -30 and -50 translation coordinates. The 5"terminal nucleotide of each form of the apo-I1 message is indicated next to the appropriate sequence listed on the right. In addition, the full length cDNAs which correspond to each form of the message are indicated next to the control (-) lane. Note that apo-I1 forms I , 2, and 3 arise from initiations at -11, +1, and +7, respectively. The A forms of mRNA correspond to processing at the upstream AG doublet and the B forms correspond to splicing at the downstream AG doublet (see Fig. 6).

minor form (light band intensity) of the message corresponds exactly to the published mRNA sequence as it was determined from an apo-I1 cDNA clone (13). The major form of the sequence, however, corresponds to the published sequence with the insertion of an additional 5'-CAG-3' (Fig. 5, arrow A ) at nucleotide -43. This sequence divergence in the mature message occurs near the splice junction formed by the joining of RNA encoded by exons I and I1 and can be explained by the presence of an unusual splice acceptor site (3' splice site)

(16) at the intron A-exon I1 border. Since this 3' splice site region contains two adjacent CAG triplets (5' . . . CAGCAG . . . 3') (15), it apparently provides alternate sites for excision of the 3' end of intron A. This is diagrammed in Fig. 6 and is discussed further below.

Quuntitation of Multiple Apo-11 mRNA Species-In order to measure the relative abundance of the mature forms of the apo-I1 message, we repeated the primer extension analysis using probe A (Figs. 1 and 2) with seven different RNA preparations each isolated from a different estrogen-stimu- lated animal. The extended products were separated by 10% denaturing polyacrylamide gel electrophoresis and the labeled fragments were visualized by autoradiography. To quantitate the radioactivity in each band, fragments were cut from the gel, solubilized in 30% hydrogen peroxide, and counted. Con- trol experiments showed that the primer extension signals behaved as a linear function of the RNA input (data not shown). The results (Table I) show that primary transcripts

79

'Y Y Y Y Y Y Y Y Y Y Y N Y A * G ~ ' Consensus 96 100 52

Y Y Y Y Y Y Y Y Y Y Y N Y A G ~ Consensus

Gm]CIAG? Apo n-A m R N A

G G A C A G I G T C T C Apo I I -B m R N A

FIG. 6. Heterogeneous splicingof apo-I1 mRNA. The 3' splice site region (16) a t the intron A-exon I1 border is shown as it would exist in the apo-I1 primary transcription product (Apo I I IO), although T has been used in place of U. The 3'4s consensus sequence (17, 40) has been aligned differently above and below the primary tran- scription product to reflect the two different splice sites which are utilized for excision of the 3' end of the intron. The numbers on top refer to the per cent Occurrence of the indicated nucleotides in the 130 3'4s regions used to formulate the consensus sequence (40). The pyrimidine (Y) track has been bracketed to indicate that the average Occurrence of Y at these positions is 79%. Numbers above the primary transcript indicate per cent Occurrence of nucleotides that do not fit the consensus when the consensus is aligned as it is at the top. Numbers listed below the primary transcript indicate per cent Occurrence of nucleotides that do not fit the consensus when the consensus is aligned as it is below the primary transcript. Note that in the lower alignment the A and G nucleotides have a 10 and 18% Occurrence individually although they have never been found in these positions as adjacent nucleotides (36). This feature of the apo-113'- SS is indicated by bracketing the AG dinucleotide and labeling it with a 0. Shown below are the two types of apo-I1 message that can be generated from a single primary transcript. Boxed nucleotides show exons (broken line is used to indicate the CAG triplet which in one case is in exon I1 and in the other case is in intron A). Introns are indicated by unboxed sequences or a solid line. The last nucleotide of an intron is designated -1 in reference to the "Discussion." N , any nucleotide.

TABLE I Relative abundance of each form of apo-II mRNA

Each form of the message was quantitated as described under "Results" and expressed as a percentage of the total. The column marked A/B is the ratio for each primary transcript ( I , 2 , and 3) of mRNA corresponding to splicing at the upstream AG doublet (A ) or the downstream AG doublet ( B ) .

Apo-I1 mRNA % total AIB

IA IB 2A 2B 3A 3B

8.7 f 1.4 2.1 4.1 f 0.7

60.4 2 2.0 2.7 22.4 f 0.4 3.6 f 0.6 2.8 1.3 & 0.4

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9934 Transcription and Processing of Apo-11 mRNA

initiated at +1 (2A, 2B) and +7 (3A, 3B) yield the two forms of mature mRNA in a ratio of 2.7:l (A:B). In contrast, primary transcripts originating at -11 ( IA, 1B) yield mature apo-I1 mRNAs in a ratio of only 2.1:1 (A:B). This result may indicate an effect of the 5’ leader sequence on the splicing mechanism or on mRNA stability.

DISCUSSION

S1 and primer extension mapping, coupled with cDNA primed dideoxy-RNA sequencing, have been used to identify six forms of apo-I1 mRNA with different 5’-untranslated leader sequences. The multiple forms of apo-I1 mRNA, dia- grammed in Fig. 5, arise due to three sites of transcription initiation on the gene as well as alternate splicing which yields two species of mature message from each primary transcript (Fig. 6). The apparent basis for this splicing heterogeneity is an unusual sequence around the intron A-exon I1 border.

To illustrate this feature of apo-I1 mRNA processing, Fig. 6 shows a comparison between the sequence around the apo- I1 3“SS and the 3”SS consensus sequence based on a com- pilation of 130 intron-exon borders (17, 39). As indicated in Fig. 6, the only strictly conserved region around the 3’-SS is the AG dinucleotide a t positions -2,-1, with respect to the splice site. Furthermore, while there is a high degree of diver- gence in the sequence between -5 and -15, the AG dinucleo- tide is virtually never found in this region (17, 39). These features of the 3’-SS consensus sequence have been used to argue that the splicing process at the 3‘ end of an intron proceeds via a “micro” scanning mechanism whereby a protein or protein-RNA (16, 40) complex binds to the (Y), region preceding the 3’-SS and scans in a 5‘- 3‘ direction until the first AG dinucleotide is encountered (39, 41).

The 3”SS region at the intron A-exon I1 border of the apo- I1 primary transcript provides an interesting test of this hypothesis since it contains two adjacent CAG triplets after the (Y), region (see Fig. 6). An initial characterization by van het Schip et al. (15) suggested that this 3”SS did, in fact, violate the consensus, since splicing was found to occur only at the downstream AG dinucleotide. This conclusion was based on a comparison of a cloned genomic apo-I1 sequence with an apo-I1 cDNA clone (13). In our present analysis, we have used dideoxy-RNA sequencing to sequence across the splice junction in the mature mRNA formed by the joining of RNA encoded by exons 1 and 11. This analysis shows that, while some splicing occurs at the downstream AG, as previ- ously reported (15), the majority occurs at the first AG dinu- cleotide following the (Yfn region. Thus, the majority of splicing events are in accord with the predictions of the consensus sequence (17, 39). This feature of apo-I1 mRNA was previously unknown because the cloned cDNA sequence (13) used to assign the intron-exon organization of the gene, corresponds to an mRNA spliced at the downstream AG dinucleotide (15). Quantitation of primer extension products corresponding to apo-I1 mRNAs spliced at either the upstream or downstream AG dinucleotide indicates a processing ratio of 2.1:l or 2.7:l (upstream:downstream) depending on the location of the cap site (Table I and “Results”).

Much interest has focused on the possible directionality of splice site selection. In many cases of alternate splicing, upstream splice sites are utilized preferentially (42-45), indi- cating a 5‘-3‘ directionality to the splicing process. Recent work by Kuhne et al. (46), however, has shown that in cases where 5‘ and 3‘ splice site regions are duplicated, it is the distal (with respect to the intron) duplicated splice sites which are preferentially utilized. This pattern of splice site selection

is not consistent with a simple 5’-3’ or a 3’-5’ scanning model. Instead Kuhne et al. have suggested that the initial binding of splice sites by splicing enzymes might be mediated by primary sequence and modulated by higher order RNA structure but that the ultimate joining of correct 3’ and 5’ splice sites is determined by the relative position of these regions as determined by the inherent structure of the RNA and/or interactions between RNP proteins. If this is the case, it is likely that the preference for the upstream AG dinucleo- tide at the apo-I1 intron A-exon I1 border is a reflection of the sequence-mediated binding of the splicing enzyme(s) since it is doubtful that the three-nucleotide difference in position of the two AG dinucleotides would place these two substrates in dramatically different structural domains with respect to the corresponding 5 ‘ 3 s . In addition, the proximity of these two 3’ splice sites probably precludes two separate enzymes or enzyme complexes binding simultaneously.

In addition to the case reported here, there are other in- stances of intron-exon borders containing AG dinucleotides separated by three nucleotides. In the case of rat prolactin, two different cDNA clones have been isolated which differ by the presence or absence of a 5’-GCA-3’ triplet at the first splice junction in the mature mRNA (47-49). Since the se- quence preceding the 3“SS at the first intron exon border is 5’ . . . TAGCAG . . .3’, it can be inferred that the two cDNAs represent mRNAs formed by intron removal after either the upstream or downstream AG dinucleotide (49). On the other hand, this has not been shown at the mRNA level, nor has the relative frequency of each splicing event been quantified, although the indication is that, as with apo-11, the upstream AG is used preferentially as the 3”SS (49, 50).

It is interesting to note that closely spliced AG dinucleotides are not alone sufficient to support alternate splicing. The 3’- SS at the intron A-exon I1 border of the gene encoding the common a subunit of the four human glycoprotein hormones contains the sequence 5’ . . . CAGGAG . . . 3’, yet the data of Fiddes and Goodman (51) indicates that splicing occurs only at the upstream AG dinucleotide. Since the 3”SS consensus specifies that the nucleotide at position -3 should be a pyrim- idine (17, 39) it is possible that the presence of a G residue in this position is sufficient to abolish or greatly diminish splic- ing at the downstream AG dinucleotide. On the other hand, there is at least one instance where a functional 3’-SS is preceded by a G residue at position -3 (39, 52).

Our results on the location of transcription initiation sites on the apo-I1 gene are in partial agreement with previous reports. Hache et al. (14) used S1 protection mapping to identify three apo-I1 mRNA precursors whose 5‘ ends map to positions -19, -9, and +7. van het Schip et al. (15) also used S1 mapping to demonstrate the existence of at least two apo- I1 mRNA precursors which map to positions -11 and +l. In each case, considerable microheterogeneity complicated inter- pretation of the mapping results. In the present study, parallel S1 and primer extension analyses were performed using intron probes which share a common 5’-labeled end. Both analyses detect three forms of apo-I1 mRNA precursors. The 5’ ends of two of these precursors map to the same position on the apo-11 gene (-11 and +I) as the two forms described by van het Schip et al. (15). The 5‘ end of the third precursor maps to position +7. The relative abundance of these precursors is 15% (-ll), 80% (+l), and 5% (+7). The abundance of the mature apo-I1 mRNAs initiated at each of three different sites is 13% (-11), 82% (+l), and5% (+7) (Table I). The similarity in these values suggests that the relative abundance of the mature mRNAs is largely a reflection of transcription and not mRNA stability.

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Transcription and Processing of Apo-II mRNA 9935

Interestingly, the presence of multiple initiation sites has been found for other chicken genes whose transcription is estrogen dependent. These include the genes encoding the major egg white proteins ovalbumin (53-55), ovomucoid (561, and lysozyme (57,58). Of these three cases, the lysozyme and ovomucoid genes bear the most similarity to apo-I1 since these genes contain separate TATA-like homologies upstream from each site or group of sites of transcription initiation (14, 15, 59). The basis for multiple promoter sites or the possible regulation of promoter selection in these systems is not yet understood.

Acknowledgments-We are grateful to R. Axel for providing us with the chicken DNA library. We would also like to thank Salvatore Mungal for technical assistance and Drs. Daniel Bogenhagen, Patrick Hearing, and Carl Palatnik for critical reading of the manuscript.

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G S Shelness and D L Williamsyields six 5'-untranslated leader sequences.

Apolipoprotein II messenger RNA. Transcriptional and splicing heterogeneity

1984, 259:9929-9935.J. Biol. Chem. 

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