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• W2014494306 abstract "Three exons in the fibronectin primary transcript are alternatively spliced in a tissue- and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3′-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric β-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context. Three exons in the fibronectin primary transcript are alternatively spliced in a tissue- and developmental stage-specific manner. One of these exons, EDA, has been shown previously by others to contain two splicing regulatory elements between 155 and 180 nucleotides downstream of the 3′-splice site: an exon splicing enhancer and a negative element. By transient expression of a chimeric β-globin/fibronectin EDA intron in COS-7 cells, we have identified two additional exonic splicing regulatory elements. RNA generated by a construct containing the first 120 nucleotides of the fibronectin EDA exon was spliced with an efficiency of approximately 50%. Deletion of most of the fibronectin EDA exon sequences resulted in a 20-fold increase in the amount of spliced RNA, indicative of an exon splicing silencer. Deletion and mutagenesis studies suggest that the fibronectin exon splicing silencer is associated with a conserved RNA secondary structure. In addition, sequences between nucleotides 93 and 118 of the EDA exon contain a non-purine-rich splicing enhancer as demonstrated by its ability to function in a heterologous context. Higher eukaryotes have evolved alternative splicing as a common means of producing several different mRNAs and polypeptides from a single primary transcript (for review, see Ref. 1Smith C.W.J. Patton J.G. Nadal-Ginard B. Annu. Rev. Genet. 1989; 23: 527-577Crossref PubMed Scopus (565) Google Scholar). Transcripts that undergo alternative splicing can produce mRNAs and proteins with different structures, properties, and functions. The utilization of alternative splice sites can be regulated to prevent the inappropriate accumulation of one or more alternative products. To date, several examples of cis-acting regulatory elements, distinct from bona fide splice sites, have been identified which modulate splicing in a diverse assortment of genes from viruses (2Staffa A. Cochrane A. Mol. Cell. Biol. 1995; 15: 4597-4605Crossref PubMed Scopus (150) Google Scholar, 3Amendt B.A. Si Z.-H. Stoltzfus C.M. Mol. Cell. Biol. 1995; 15: 4606-4615Crossref PubMed Scopus (122) Google Scholar, 4Amendt B.A. Hesslein D. Chang L.J. Stoltzfus C.M. Mol. Cell. Biol. 1994; 14: 3960-3970Crossref PubMed Scopus (125) Google Scholar, 5Amendt B.A. Simpson S.B. Stoltzfus C.M. J. Virol. 1995; 69: 5068-5076Crossref PubMed Google Scholar, 6McNally M.T. Gontarek R.R. Beemon K. Virology. 1991; 185: 99-108Crossref PubMed Scopus (62) Google Scholar, 7Nemeroff M.E. Utans U. Kramer A. Krug R.M. Mol. Cell. Biol. 1992; 12: 962-970Crossref PubMed Scopus (41) Google Scholar, 8Zheng Z.M. He P.J. Baker C.C. J. Virol. 1996; 70: 4691-4699Crossref PubMed Google Scholar, 9Pintel D.J. Gersappe A. 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The fibronectin gene is a classical example of a gene that undergoes alternative splicing (34Hynes R.O. Rich A. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar, 35Sharp P.A. Cell. 1994; 77: 805-815Abstract Full Text PDF PubMed Scopus (446) Google Scholar). The fibronectin gene is evolutionarily conserved and is predicted to yield up to 20 different mRNAs in human cells. The generation of this remarkable number of different mRNAs, and consequently polypeptides, is made possible by alternative splicing in three different coding regions of the fibronectin primary transcript (for review, see Refs. 36Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar and 37ffrench-Constant C. Exp. Cell Res. 1995; 221: 261-271Crossref PubMed Scopus (166) Google Scholar). One of the alternatively spliced regions encompasses the EDA (also referred to as EIIIA or EDI) exon. This exon is excluded selectively in fibronectin mRNAs produced by hepatocytes, but it is included to various extents by other cell types (Fig. 1 A). EDA exon splicing is also subject to developmental regulation as EDA exon inclusion generally declines with age and differentiation (36Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar, 37ffrench-Constant C. Exp. Cell Res. 1995; 221: 261-271Crossref PubMed Scopus (166) Google Scholar). In addition to tissue-specific and developmental regulation, inclusion of the EDA exon is modulated during the process of tissue repair and in certain diseases. For these reasons, regulation of fibronectin EDA exon splicing has been the subject of several studies. In fact, the EDA exon was the first exon with which it was demonstrated that proper alternative splicing could occur in the context of transfected minigenes (38Vibe-Pedersen K. Kornblihtt A.R. Baralle F.E. EMBO J. 1984; 3: 2511-2516Crossref PubMed Scopus (70) Google Scholar). Using the same experimental approach, it was determined subsequently that the central 81 nucleotides of the 270-nucleotide EDA exon were required for its inclusion into mRNA (31Mardon H.J. Sebastio G. Baralle F.E. Nucleic Acids Res. 1987; 15: 7725-7733Crossref PubMed Scopus (89) Google Scholar). Using a chimeric adenovirus/fibronectin EDA intron substrate in an in vitrosplicing system, Lavigueur et al. (32Lavigueur A. La Branche H. Kornblihtt A.R. Chabot B. Genes Dev. 1993; 7: 2405-2417Crossref PubMed Scopus (272) Google Scholar) further mapped the element required for EDA exon inclusion to a 9-nucleotide purine-rich motif (5′-GAAGAAGAC-3′). Caputi et al. (33Caputi M. Casari G. Guenzi S. Tagliabue R. Sidoli A. Melo C.A. Baralle F.E. Nucleic Acids Res. 1994; 22: 1018-1022Crossref PubMed Scopus (137) Google Scholar) identified the same purine-rich sequence as a positive cis-acting element by transfection experiments. Similar exonic purine-rich motifs have been demonstrated to stimulate splicing (39Tanaka K. Watakabe A. Shimura Y. Mol. Cell. Biol. 1994; 14: 1347-1354Crossref PubMed Scopus (206) Google Scholar, 40Xu R. Teng J. Cooper T.A. Mol. Cell. Biol. 1993; 13: 3360-3374Google Scholar, 41Sun Q. Mayeda A. Hampson R.K. Krainer A.R. Rottman F.M. Genes Dev. 1993; 7: 2598-2608Crossref PubMed Scopus (245) Google Scholar) and have been termed exon recognition sequences or exon splicing enhancers (ESEs). 1The abbreviations used are: ESE(s), exon splicing enhancer(s); 5′ss, 5′-splice site; 3′ss, 3′-splice site; ESS, exon splicing silencer; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; bp, base pair(s); HIV-1, human immunodeficiency virus type 1. 1The abbreviations used are: ESE(s), exon splicing enhancer(s); 5′ss, 5′-splice site; 3′ss, 3′-splice site; ESS, exon splicing silencer; PCR, polymerase chain reaction; CAT, chloramphenicol acetyltransferase; bp, base pair(s); HIV-1, human immunodeficiency virus type 1. A negative element (5′-CAAGG-3′) involved in down-regulation of EDA exon inclusion was also identified within the EDA exon just downstream of the ESE (33Caputi M. Casari G. Guenzi S. Tagliabue R. Sidoli A. Melo C.A. Baralle F.E. Nucleic Acids Res. 1994; 22: 1018-1022Crossref PubMed Scopus (137) Google Scholar). Although the ESE within the EDA exon has been demonstrated (32Lavigueur A. La Branche H. Kornblihtt A.R. Chabot B. Genes Dev. 1993; 7: 2405-2417Crossref PubMed Scopus (272) Google Scholar) to interact with splicing factors belonging to a family of serine/arginine-rich RNA-binding proteins collectively termed SR proteins (42Fu X.-D. RNA. 1995; 1: 663-680PubMed Google Scholar), it is not yet known if trans-acting factors interact with the negative element. Using a chimeric intron composed of sequences flanking the 5′-splice site (5′ss) of the first intron of human β-globin and sequences flanking the 3′-splice site (3′ss) of the human fibronectin EDA exon (2Staffa A. Cochrane A. Mol. Cell. Biol. 1995; 15: 4597-4605Crossref PubMed Scopus (150) Google Scholar), we observed that deletion of most of the fibronectin EDA exon sequences resulted in a dramatic increase in splicing efficiency. We have carried out mapping experiments to characterize this novel exon splicing silencer (ESS) element. Closer examination of the ESS revealed an adjacent positive element that is able to stimulate splicing when placed downstream of a heterologous intron. This novel ESS and adjacent positive element represent the third and fourth splicing regulatory elements to be identified within the 270-nucleotide EDA exon. The presence of so many different regulatory elements is of particular interest because they may explain the complex, dynamic splicing pattern exhibited by the fibronectin EDA exon. All plasmids are derivatives of pSVβFN– (2Staffa A. Cochrane A. Mol. Cell. Biol. 1995; 15: 4597-4605Crossref PubMed Scopus (150) Google Scholar). Truncation of fibronectin exon sequences (accession no.X07718) was carried out by PCR using the sense primer FN3′ss, 5′-CGT CGA CAA AGA AAA TGG TAT CTG C-3′ (nucleotides 1146–1164) and the following antisense primers: Δ, 5′-CCC CCG GAT CCA ATG CCA GTC CTT TAG GG-3′ (nucleotides 1255–1272); Δ1, 5′-CGG GAT CCC ACC CTG TAC CTG GA-3′ (nucleotides 1331–1345); Δ2, 5′-CGG GAT CCA ACT TGC CCC TGT GG-3′ (nucleotides 1316–1330); Δ3, 5′-CGG GAT CCG CTT TCC CAA GCA AT-3′ (nucleotides 1301–1315); and Δ4, 5′-CGG GAT CCT TTG ATG GAA TCG AC-3′ (nucleotides 1286–1300) to generate the respective constructs. The mod series of fibronectin exon mutations was carried out by PCR-mediated site-directed mutagenesis as described previously (43Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar) using the FN3′ss sense primer and the antisense primer CATR1 (described below) along with the following mutagenic primers: mod1, 5′-CAC AGG GGC AAGatcTAC AGG GTG ACC-3′ (nucleotides 1317–1348); mod2, 5′-CAT CAA AAT TGCagatct CAC AGG GGC AAG-3′ (nucleotides 1294–1328); and mod3, 5′-CAC TGA TGT GGAgatcTC AAA ATT GCT T-3′ (nucleotides 1273–1307) to generate the respective constructs. Lowercase letters indicate nucleotides introduced to create a unique BglII restriction enzyme site (underlined). The single stem-loop 1 mutation (SL1B) was generated in a similar fashion using the mutagenic primer SL1B, 5′-TCA CTG ATG TGG tac TCG ATT CCA T-3′ (nucleotides 1272–1296), except that it was introduced in the context of the Δ1 truncation. The single stem-loop 2 mutation (SL2B) was generated in the context of the Δ1 truncation by PCR using FN3′ss sense primer and the SL2B antisense primer 5-CCG GAT CCC ACC CTG TAC Cac cAA ACT TGC CCC TGT G-3′ (nucleotides 1317–1345). The double stem-loop 2 mutation (SL2TB) was derived from pSVβFNSL2B by PCR-mediated site-directed mutagenesis using the mutagenic primer SL2T, 5′-GAT TCC ATC AAA ATT GCT acc GAA AGC CCA CAG-3′ (nucleotides 1289–1321). The double stem-loop 1 mutation (SL1TB) was generated as follows. The PCR product generated using the FN3′ss/SL1T primer pair was gel purified and mixed with an equimolar amount of gel purified PCR product generated with the SL1T, 5′-CCA CAT CAG TGA tac CCA GTC CTT T-3′ (nucleotides 1259–1283)/CATR1 (described below) primer pair. One end of each of these two DNA fragments contains a 12-bp overlap flanked by either the SL1B or SL1T mutation. By virtue of this 12-bp overlap, the two fragments were annealed and elongated by 10 cycles of low stringency PCR (25 °C annealing step) and then amplified by 25 cycles of normal stringency PCR (55 °C annealing step) using the FN3′ss/CATR1 primer pair. Approximately 75% of clones sequenced contained the desired double mutation. The SalI-BamHI fragments of truncated or mutated PCR products were reinserted into the SalI and BamHI sites of pSVβFN–. The plasmid pSVCBSB has been described previously (43Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar). Its derivatives, containing fibronectin EDA exon sequences in the sense (pSVCBSBFs) or antisense (pSVCBSBFas) orientation, were generated by inserting the 30-bp BglII-BamHI fragment of pSVβFNmod1 into the unique BamHI site of pSVCBSB. The +1C, Δ5′ss, and ΔAC mutations were introduced in the context of pSVCBSBFs by PCR using the sense primer βPCR1, 5′-CTT AAG TTG GTG GTG AGG-3′ (43Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar), in conjunction with the following antisense primers: +1C, 5′-CGG ATC CGG TCA GGG CTA GAG TAG GTC AgC CTG TAG ATC CG-3′ (nucleotides 1337–1362); Δ5′ss, 5′-CCG GAT CCG GTC AGG GCT CGA GTA GGT TGT AGA TCC GGT CTG-3′ (nucleotides 1337–1340, 1346–1362); and ΔAC, 5′-CCG GAT CCG GTC ACC CTG TAG ATC CG-3′ (nucleotides 1337–1348). The SalI-BamHI fragments of the resulting PCR products were reinserted into the SalI and BamHI sites of pSVCBSB. COS-7 cells (ATCC CRL1651) were transfected as described previously (44Cullen B.R. Methods Enzymol. 1988; 152: 684-704Crossref Scopus (662) Google Scholar) with 5 μg of plasmid DNA/100-mm Petri dish. 48 h post-transfection, cells were washed once with ice-cold phosphate-buffered saline, lysed directly on the culture dish by the addition of 1 ml of 4 m guanidine thiocyanate, 25 mm sodium citrate (pH 7.0), 0.5% sarkosyl, and 0.1 m β-mercaptoethanol, and total RNA was isolated as described previously (45Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62909) Google Scholar). When actinomycin D was used, the drug was added to the culture medium 48 h post-transfection at a final concentration of 5 μg/ml, and total RNA was extracted as described above at the indicated times. To generate probes for S1 nuclease analysis, the XhoI-SalI fragment of each construct was deleted, and the resulting plasmids were linearized with PvuI. Linearized plasmids were used as templates for primer extension using the 5′-end labeled antisense primer CATR1, 5′-CGG AAT TCC GGA TGA GCA TT-3′, which overlaps the EcoRI restriction endonuclease site of the chloramphenicol acetyltransferase (CAT) open reading frame. 50 fmol of PvuI-linearized template DNA and 1 pmol of 5′-end-labeled CATR1 primer were subjected to 15 thermal cycles (1 min at 94 °C, 30 s at 55 °C, and 1.5 min at 72 °C) with 2 units of Taq DNA polymerase in a 20-μl reaction. Free CATR1 primer was removed by agarose gel purification of the extended primer. S1 nuclease protection assays were carried out as described previously (43Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar) using 10 μg of total RNA and 5–10 × 104 cpm (approximately 50–100 fmol) of the appropriate probe. S1-resistant probe DNA fragments were subjected to electrophoresis on 4% polyacrylamide, 8 m urea gels and subsequently visualized and quantitated using a PhosphorImager (Molecular Dynamics). All cited ratios of spliced to unspliced RNAs are averages of results obtained in at least three independent experiments. In previous studies, our laboratory generated a chimeric gene composed of sequences flanking the 5′ss of the first intron of the human β-globin gene and sequences flanking the 3′ss upstream of the EDA exon of the human fibronectin gene (2Staffa A. Cochrane A. Mol. Cell. Biol. 1995; 15: 4597-4605Crossref PubMed Scopus (150) Google Scholar). The fibronectin sequences within the chimeric construct include 99 nucleotides of the intron and the first 118 nucleotides of the EDA exon (Fig.1 B). The previously characterized purine-rich ESE and the adjacent negative element, which are located between 155 and 180 nucleotides downstream of the EDA 3′ss, are not present in pSVβFN– and thus do not contribute to the level of splicing observed. S1 analysis of RNA isolated from transfected COS-7 cells showed that splicing of pSVβFN– RNA is relatively inefficient and results in the accumulation of roughly equal amounts of spliced and unspliced RNA (Fig. 1 C, lane 1). In an attempt to study the positional effect of a heterologous ESS element, we generated a truncated form of the EDA exon (designated pSVβFNΔ; see Fig. 2) in which sequences from +28 to +118 were deleted. Deletion of these sequences resulted in a 20-fold increase in the ratio of spliced to unspliced (S:U) RNA (Fig. 1 C, lane 2). This finding indicates that a splicing silencer element may exist within this region of the fibronectin EDA exon and that the EDA 3′ss may be intrinsically efficient. To map the fibronectin ESS element, we generated sequential 3′-truncations of the EDA exon sequences (Fig. 2). S1 analysis of RNA from COS-7 cells transfected with pSVβFNΔ1, Δ2, Δ3, or Δ4 (Fig.3) revealed that, with the exception of Δ1 (lane 2), splicing efficiency steadily increased as fibronectin EDA exon sequences were progressively shortened (lanes 3–5); the ratio of spliced to unspliced RNA approached that obtained with pSVβFNΔ. The Δ1 truncation defines the 3′-boundary of the fibronectin ESS as nucleotide +101 relative to the EDA 3′ss. The correlation between the size of the truncation and the splicing efficiency observed with Δ2, Δ3, and Δ4 suggests that the fibronectin ESS may be comprised of multiple smaller elements dispersed within the first 100 nucleotides of the EDA exon or that its function is dependent upon the stability of a secondary structure. However, based on the effect of these 3′-deletions alone, we could not exclude the possibility that the progressive increase in splicing is caused by the positioning of CAT sequences closer to the EDA 3′ss and not by disruption of an ESS within the EDA exon. To test this possibility, we used site-directed mutagenesis to introduce small internal deletions in the EDA exon within the context of pSVβFN– (Fig. 2). The net effect of these internal modifications is a 5-nucleotide deletion in pSVβFNmod1 and pSVβFNmod2 and a 7-nucleotide deletion in pSVβFNmod3. All three mutations resulted in a moderate (4–8-fold) increase in splicing efficiency (Fig.4). These results suggest that the effects seen in Fig. 3 are caused by deletion of a splicing silencer element between nucleotides +28 and +101 rather than by movement of CAT sequences closer to the EDA 3′ss. To verify that the alteration in the spliced:unspliced RNA ratio is not attributable to differences in RNA stability, the half-life of the spliced and unspliced RNAs of several constructs was examined. As indicated in Fig.5, all three constructs tested had half-lives of approximately 6 h for spliced RNA (filled circles) and 2–4 h for unspliced RNA (empty circles). Thus, no apparent difference in the stability of spliced RNA was observed among pSVβFN–, pSVβFNmod3, and pSVβFNΔ4. Therefore, the marked increase in accumulation of spliced RNA cannot be attributed to a longer half-life. The small decrease in half-life observed for the unspliced RNA of pSVβFNΔ4 compared with that of pSVβFN– or pSVβFNmod3 is consistent with more efficient splicing kinetics.Figure 5Increased accumulation of spliced RNA is not caused by changes in RNA stability. COS-7 cells transfected with pSVβFN–, pSVβFNΔ4, or pSVβFNmod3 were treated with 5 μg/ml actinomycin D (Act D). Total RNA isolated at 0, 3, 6, and 9 h after the addition of the drug was analyzed by S1 nuclease protection. Unspliced and spliced RNA was quantitated and expressed as the percentage of the respective RNA (spliced or unspliced) at 0 h detected at the indicated time. Spliced RNA is indicated by filled circles and unspliced RNA by empty circles. Dotted, solid, and dashed lines represent pSVβFN–, pSVβFNΔ4, and pSVβFNmod3, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The complete abrogation of splicing observed with the pSVβFNΔ1 construct suggests that the sequences deleted in pSVβFNΔ1 (nucleotides 102–118) harbor a splicing enhancer element. However, if that is the case, it differs from “classical” ESE elements (39Tanaka K. Watakabe A. Shimura Y. Mol. Cell. Biol. 1994; 14: 1347-1354Crossref PubMed Scopus (206) Google Scholar, 40Xu R. Teng J. Cooper T.A. Mol. Cell. Biol. 1993; 13: 3360-3374Google Scholar, 41Sun Q. Mayeda A. Hampson R.K. Krainer A.R. Rottman F.M. Genes Dev. 1993; 7: 2598-2608Crossref PubMed Scopus (245) Google Scholar) in that it is not purine-rich. To test if these EDA exon sequences could stimulate splicing when placed in a heterologous context, nucleotides 93–118 of the EDA exon were inserted into pSVCBSB (Fig. 6 A) downstream of a chimeric β-globin/HIV-1 intron. The pSVCBSB chimeric intron is not spliced (43Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar) since critical HIV-1 exon splicing regulatory elements have been deleted (2Staffa A. Cochrane A. Mol. Cell. Biol. 1995; 15: 4597-4605Crossref PubMed Scopus (150) Google Scholar). We have demonstrated previously that splicing of this intron can be stimulated significantly by several classical purine-rich ESEs, including the ESE within the fibronectin EDA exon. Consistent with our previously published results (43Staffa A. Cochrane A. J. Virol. 1994; 68: 3071-3079Crossref PubMed Google Scholar), only trace amounts of spliced RNA were detected with pSVCBSB (Fig. 6 B,lane 1). Insertion of nucleotides 93–118 of the EDA exon in the sense orientation (pSVCBSBFs) stimulated splicing at least 25-fold (Fig. 6 B, lane 2). Conversely, insertion of the same EDA sequences in the antisense orientation did not have a stimulatory effect on splicing (Fig. 6 B, lane 3). The region associated with enhancer activity contains the sequence 5′-AGGGTGACC-3′, which bears homology (a 6 out of 9 match; underlined) to the 5′ss consensus sequence, 5′-(C/A)AGGT(A/G)AGT-3′. One model to explain how this novel enhancer functions is that this pseudo-5′ss element recruits U1 snRNP to stimulate recognition of the EDA 3′ss much like authentic 5′-splice sites have been demonstrated to stimulate exon recognition (46Robberson B.L. Cote G.J. Berget S.M. Mol. Cell. Biol. 1990; 10: 84-94Crossref PubMed Scopus (546) Google Scholar, 47Hoffman B.E. Grabowski P.J. Genes Dev. 1992; 6: 2554-2568Crossref PubMed Scopus (163) Google Scholar). To examine this possibility, we introduced a point mutation that converted the critical GT dinucleotide of the pseudo-5′ss to CT (designated +1C in Fig. 6 C), deleted the majority of the pseudo-5′ss (Fig.6 C, Δ5′ss), or deleted the A/C-rich region immediately downstream of the pseudo-5′ss (Fig. 6 C, ΔAC). Analysis of the effect of these mutations revealed that none of the mutations tested resulted in loss of enhancer function. Rather, in all cases the mutation resulted in a further enhancement in the ratio of spliced to unspliced RNA (Fig. 6 D, compare lanes 3–5 with lane 2). Therefore, the splicing enhancer activity associated with this non-purine-rich sequence cannot be attributed solely to the 5′ss-like element. The relatively large size of the fibronectin ESS prompted us to examine its ability to form a secondary structure. The 118 nucleotides of the EDA exon present in pSVβFN– are predicted to form three stem-loops (Fig.7 A). Each of the two stem-loops closest to the EDA 3′ss contains a hairpin loop of 4 nucleotides and an internal loop that interrupts the 13-bp stem. The third and most distal stem-loop is predicted to consist of a stem of only 6 bp and a relatively large loop of 11 nucleotides. The fibronectin gene and its alternative splicing patterns are highly conserved among human, rat, and chicken (36Kornblihtt A.R. Pesce C.G. Alonso C.R. Cramer P. Srebrow A. Werbajh S. Muro A.F. FASEB J. 1996; 10: 248-257Crossref PubMed Scopus (172) Google Scholar, 37ffrench-Constant C. Exp. Cell Res. 1995; 221: 261-271Crossref PubMed Scopus (166) Google Scholar). Therefore, we examined whether the first 118 nucleotides of the EDA exon of other species retained the potential to form a secondary structure similar to that in Fig. 7 A. Fibronectin sequences of different species were obtained from sequence data bases and aligned (Fig.7 B). Compared with the human fibronectin sequence, the EDA exons from fibronectin of other mammals such as dog, rat, and mouse differ by only a few nucleotides, whereas those of chicken, newt, and frog are slightly more divergent. As expected, most of these nucleotide substitutions are silent at the amino acid level. The interesting observation is the occurrence of covariance and conservative substitutions. The region comprising step-loop 1 is completely conserved among human, dog, and rat fibronectin sequences. Chicken, newt, and frog fibronectin sequences contain several substitutions that do not compromise the ability of this region to form step-loop 1. An example of covariance is seen in chicken fibronectin involving positions +23 and +44. As depicted in Fig. 7 A, the nucleotides at these two positions are predicted to form a G-C bp in stem-loop 1. In chicken fibronectin, the nucleotide at +23 is replaced by an A, and a compensatory C → T substitution occurs at nucleotide +44, thus maintaining the ability to form a canonical Watson-Crick bp. Several examples of conservative substitutions involving putative base pairing between G and U residues are also observed. In both chicken and frog, a G-U bp in stem-loop 1 is converted to a canonical A-U bp by a G → A substitution at position +24. Examples in which canonical Watson-Crick bp are replaced by a wobble bp are also observed. In frog, a U-A bp is converted to a U-G bp by an A → G substitution at +40, and a G-C bp is converted to a G-U bp by a C → T substitution at +44. Taken together, these examples of covariance and conservative nucleotide substitutions support the notion that the stem-loop 1 secondary structure depicted in Fig. 7 Ais absolutely conserved and is indicative of selective pressure for maintenance of this secondary structure in addition to the protein-coding potential. Compared with stem-loop 1, there are only a few examples of covariance or conservative substitutions in the region comprising the putative stem-loop 2." @default.
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• W2014494306 title "Novel Exonic Elements That Modulate Splicing of the Human Fibronectin EDA Exon" @default.
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