Elements of the rat tropoelastin gene associated with alternative splicing

Multiple isoforms of tropoelastin, the soluble precursor of elastin, are the products of translation of splice-variant mRNAs derived from the single-copy tropoelastin gene. Previous data had demonstrated DNA sequence heterogeneity in three domains of rat tropoelastin mRNA, indicating alternative splicing of several exons of the rat tropoelastin gene. Rat tropoelastin genomic clones encompassing the sites of alternative splicing were isolated and sequenced. Two sites of alternative splicing identified in rat tropoelastin mRNA sequences corresponded to exons 13-15 and exon 33 of the rat tropoelastin gene. Furthermore, the variable inclusion of an alanine codon in exon 16 resulted from two functional acceptor sites separated by three nucleotides. DNA sequences flanking exons subject to alternative splicing were analyzed. These exons contained splicing signals that differed from consensus sequences and from splicing signals of constitutively spliced exons. Introns immediately 5' of exons 14 and 33, for example, lacked typical polypyrimidine tracts and had weak, overlapping branch point sequences. Further, a region of secondary structure encompassing the acceptor site of exon 13 may influence alternative splicing of this exon. These results demonstrate that multiple cis-acting sequence elements may contribute to alternative splicing of rat tropoelastin pre-mRNA.     

Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF³⁵-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.     

用支持向量机预测人类基因5’／3’选择性剪切位点

选择性剪切是调解基因表达的重要机制.识别选择性剪切位点是后基因组时代的一个重要工作.本文从最新的EBI人类基因选择性剪切数据库中,选取5＇/3＇选择性剪切位点作为正集,选取在剪切位点附近的假剪切位点作为负集,并把所有的选择性剪切位点和假剪切位点随机分成训练集和测试集.本文选用的预测选择性剪切位点的方法是基于位置权重矩阵和离散增量的支持向量机方法.此方法仅基于训练集,以不同位点的单碱基概率和序列片断的三联体频数作为信息参数,利用位置权重矩阵和离散增量算法结合支持向量机,得到了选择性供体位点和受体位点的分类器,并用此分类器对测试集中的选择性供体位点和受体位点进行预测.对独立测试集中的选择性供体位点和选择性受体位点的预测成功率分别为88.74%和90.86%,特异性分别为85.62%和81.19%.本文预测选择性剪切位点的方法成功率高于其它选择性剪切位点预测方法预测成功率,此预测方法进一步提高了对选择性剪切位点的理论预测能力.     

An exonic splicing enhancer offsets the atypical GU-rich 3' splice site of human apolipoprotein A-II exon 3

Human apolipoprotein A-II (apoA-II) intron 2/exon 3 junction shows a peculiar tract of alternating pyrimidines and purines (GU tract) that makes the acceptor site deviate significantly from the consensus. However, apoA-II exon 3 is constitutively included in mRNA. We have studied this unusual exon definition by creating a construct with the genomic fragment encompassing the whole gene from apoA-II and its regulatory regions. Transient transfections in Hep3B cells have shown that deletion or replacement of the GU repeats at the 3' splice site resulted in a decrease of apoA-II exon 3 inclusion, indicating a possible role of the GU tract in splicing. However, a 3' splice site composed of the GU tract in heterologous context, such as the extra domain A of human fibronectin or cystic fibrosis transmembrane conductance regulator exon 9, resulted in total skipping of the exons. Next, we identified the exonic cis-acting elements that may affect the splicing efficiency of apoA-II exon 3 and found that the region spanning from nucleotide 87 to 113 of human apoA-II exon 3 is essential for its inclusion in the mRNA. Overlapping deletions and point mutations (between nucleotides 91 and 102) precisely defined an exonic splicing enhancer (ESEwt). UV cross-linking assays followed by immunoprecipitation with anti-SR protein monoclonal antibodies showed that ESEwt, but not mutated ESE RNA, was able to bind both alternative splicing factor/splicing factor 2 and SC35. Furthermore, overexpression of both splicing factors enhanced exon 3 inclusion. These results show that this protein-ESE interaction is able to promote the incorporation of exon 3 in mRNA and suggest that they can rescue the splicing despite the noncanonical 3' splice site.     

人类基因组盒式外显子和内含子保留的可变剪接位点预测

信使RNA的可变剪接是真核生物有别于原核生物的基本特征之一,信使RNA前体的可变剪接极大地丰富了高等真核生物蛋白质的多样性,并与生物体的组织特异性密切相关。文章对人类盒式外显子和内含子保留的一些基本特征进行了统计;根据剪接位点附近的单碱基、碱基二联体和三联体的保守性等特征,利用基于多样性指标的二次判别法,对盒式外显子和内含子保留的供体端和受体端可变剪接位点进行了预测。交叉检验结果表明,盒式外显子供体端和受体端的识别精度分别达到93％、84％以上的水平;内含子保留供体端和受体端的识别精度分别达到89％、81％以上的水平。     

A large inverted repeat sequence overlaps two acceptor splice sites in adenovirus

The distribution of nucleotide sequences resembling functional sites for mRNA splicing was examined by computer-directed searches in order to determine what factors may influence splice site selection in nuclear precursors. In particular, the distribution of large potentially stable hairpin structures or regions of extensive dyad symmetry was studied in adenovirus sequences. One region, spanning 106 nucleotides, was found at 66.4 map units, overlapping back-to-back acceptor sites for two mRNA molecules, those coding for the 100K protein and the 72K DNA binding protein, which are transcribed from opposite strands. This region displays exceptional dyad symmetry and is potentially capable of forming a single, highly stable hairpin when transcribed. It seems likely that the secondary structure as well as the primary structure of RNA plays a role in determining the correct splicing of these mRNA molecules.     

Influences of separation and adjacent sequences on the use of alternative 5' splice sites.

Single nucleotide changes to the sequence between two alternative 5' splice sites, separated by 25 nucleotides in a beta-globin gene derivative, caused substantial shifts in pre-mRNA splicing preferences, both in vivo and in vitro. An activating sequence for splicing was located. Models for the recognition by U1 small nuclear ribonucleoproteins (snRNPs) of competing 5' splice sites were tested by altering the distance separating the two sites. Use of the upstream splice site declined sharply when it was separated from the downstream (natural) site by distances of 40 nucleotides or more. This effect was reversed in vivo, but not in vitro, by altering the upstream sequence to that of a consensus 5' splice site sequence. Dilution of an extract used for splicing in vitro shifted preferences when the sites were close towards the downstream site. We conclude that the mechanism of selection depends on the distance apart of the potential splice sites and that with close sites steric interference between factors bound to both sites may impede splicing and affect splicing preferences.     

Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence.     

Assessing the fraction of short-distance tandem splice sites under purifying selection

Many alternative splice events result in subtle mRNA changes, and most of them occur at short-distance tandem donor and acceptor sites. The splicing mechanism of such tandem sites likely involves the stochastic selection of either splice site. While tandem splice events are frequent, it is unknown how many are functionally important. Here, we use phylogenetic conservation to address this question, focusing on tandems with a distance of 3-9 nucleotides. We show that previous contradicting results on whether alternative or constitutive tandem motifs are more conserved between species can be explained by a statistical paradox (Simpson's paradox). Applying methods that take biases into account, we found higher conservation of alternative tandems in mouse, dog, and even chicken, zebrafish, and Fugu genomes. We estimated a lower bound for the number of alternative sites that are under purifying (negative) selection. While the absolute number of conserved tandem motifs decreases with the evolutionary distance, the fraction under selection increases. Interestingly, a number of frameshifting tandems are under selection, suggesting a role in regulating mRNA and protein levels via nonsense-mediated decay (NMD). An analysis of the intronic flanks shows that purifying selection also acts on the intronic sequence. We propose that stochastic splice site selection can be an advantageous mechanism that allows constant splice variant ratios in situations where a deviation in this ratio is deleterious.     

Splice site consensus sequences are preferentially accessible to nucleases in isolated adenovirus RNA.

The conformation of RNA sequences spanning five 3' splice sites and two 5' splice sites in adenovirus mRNA was probed by partial digestion with single-strand specific nucleases. Although cleavage of nucleotides near both 3' and 5' splice sites was observed, most striking was the preferential digestion of sequences near the 3' splice site. At each 3' splice site a region of very strong cleavage is observed at low concentrations of enzyme near the splice site consensus sequence or the upstream branch point consensus sequence. Additional sites of moderately strong cutting near the branch point consensus sequence were observed in those sequences where the splice site was the preferred target. Since recognition of the 3' splice site and branch site appear to be early events in mRNA splicing these observations may indicate that the local conformation of the splice site sequences may play a direct or indirect role in enhancing the accessibility of sequences important for splicing.     

The different intron 2 species excised in vivo from the E2A premRNA of adenovirus-2: an approach to analyse alternative splicing.

In the early period of cellular infection by adenovirus 2, the E2A region gives rise to 2 major mRNA species of 2.0 and 2.3 kilobases, formed by alternative excisions of intron 2 (Gattoni et al., 1986, J. Mol. Biol. 187, 379-307). We have analysed the excision pathways of this intron. Two major intron species of 626 and 337 nucleotides, generated by the use of 2 consensus 3' splicing sites and a minor intron species of 520 nucleotides, generated by the use of another weaker 3' splicing site, are identified, the 3 species sharing a common 5' splicing site. They are detected predominantly in the lariat form. For the 2 major species we analyzed, the branched nucleotides are localized at consensus branching sequences, 26 or 25 nucleotides upstream from the 3' terminal AG. Our results confirm that the first reactions of cleavage at the 5' end of introns and branching occur in vivo as described in in vitro systems. The second predominant form of intron 2 is the linear segment, whereas the nicked lariat form which is very minor, might not be a genuine product of in vivo splicing. All intron 2 molecules show practically intact 5' and 3' terminal sequences, indicating that they are well protected against nuclease attack throughout their life. Therefore, these results indicate that the primary reaction following the excision of the lariat intron is debranching. In addition, the existence of a potential 5' splicing site contiguous to the major internal 3' splicing site raised the possibility of an elimination of the major 626 nucleotide intron in 2 cycles of excision. However, we demonstrate that intron 2 is systematically excised by a one cycle process, which is likely to represent the general rule for the production of correctly spliced mRNA.     

Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus.

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.