基于RNA-Seq数据识别果蝇剪接位点和可变剪接事件

完整基因结构的预测是当前生命科学研究的一个重要基础课题,其中一个关键环节是剪接位点和各种可变剪接事件的精确识别.基于转录组测序（RNA-seq）数据,识别剪接位点和可变剪接事件是近几年随着新一代测序技术发展起来的新技术策略和方法.本工作基于黑腹果蝇睾丸RNA-seq数据,使用TopHat软件成功识别出39718个果蝇剪接位点,其中有10584个新剪接位点.同时,基于剪接位点的不同组合,针对各类型可变剪接特征开发出计算识别算法,成功识别了8477个可变剪接事件（其中新识别的可变剪接事件3922个）,包括可变供体位点、可变受体位点、内含子保留和外显子缺失4种类型.RT-PCR实验验证了2个果蝇基因上新识别的可变剪接事件,发现了全新的剪接异构体.进一步表明,RNA-seq数据可有效应用于识别剪接位点和可变剪接事件,为深入揭示剪接机制及可变剪接生物学功能提供新思路和新手段.     

A clean data set of EST-confirmed splice sites from Homo sapiens and standards for clean-up procedures.

A clean data set of verified splice sites from Homo sapiens are reported as well as the standards used for the clean-up procedure. The sites were validated by: (i) standard cleaning procedures such as requiring consistency in the annotation of the gene structural elements, completeness of the coding regions and elimination of redundant sequences; (ii) clustering by decision trees coupled with analysis of ClustalW alignments of the translated protein sequence with homologous proteins from SWISS-PROT; (iii) matching against human EST sequences. The sites are categorised as: (i) donor sites, a set of 619 EST-confirmed donor sites, for which 138 are either the sites or the regions around the sites involved in alternative splice events; (ii) acceptor sites, a set of 623 EST-confirmed acceptor sites, for which 144 are either the sites or the regions around the sites are involved in alternative splice events; (iii) genuine splice sites, a set of 392 splice sites wherein both the donor and acceptor sites had EST confirmation and were not involved in any alternative splicing; (iv) alternative splice sites, a set of 209 splice sites wherein both the donor and acceptor sites had EST confirmation and the sites or the regions around them were involved in alternative splicing. A set of nucleotide regions that can be used to generate a control set of false splice sites that have a high confidence of being non-functional are also reported.     

Extent and diversity of human alternative splicing established by complementary database annotation and microarray analysis

Alternative splicing generates functional diversity in higher organisms through alternative first and last exons, skipped and included exons, intron retentions and alternative donor, and acceptor sites. In large-scale microarray studies in humans and the mouse, emphasis so far has been placed on exon-skip events, leaving the prevalence and importance of other splice types largely unexplored. Using a new human splice variant database and a genome-wide microarray to probes thousands of splice events of each type, we measured differential expression of splice types across six pair of diverse cell lines and validated the database annotation process. Results suggest that splicing in humans is more complex than simple exon-skip events, which account for a minority of splicing differences. The relative frequency of differential expression of the splice types correlates with what is found by our annotation efforts. In conclusion, alternative splicing in human cells is considerably more complex than the canonical example of the exon skip. The complementary approaches of genome-wide annotation of alternative splicing in human and design of genome-wide splicing microarrays to measure differential splicing in biological samples provide a powerful high-throughput tool to study the role of alternative splicing in human biology.     

Alternative splicing regulation at tandem 3' splice sites

Alternative splicing (AS) constitutes a major mechanism creating protein diversity in humans. Previous bioinformatics studies based on expressed sequence tag and mRNA data have identified many AS events that are conserved between humans and mice. Of these events, ~25% are related to alternative choices of 3′ and 5′ splice sites. Surprisingly, half of all these events involve 3′ splice sites that are exactly 3 nt apart. These tandem 3′ splice sites result from the presence of the NAGNAG motif at the acceptor splice site, recently reported to be widely spread in the human genome. Although the NAGNAG motif is common in human genes, only a small subset of sites with this motif is confirmed to be involved in AS. We examined the NAGNAG motifs and observed specific features such as high sequence conservation of the motif, high conservation of ~30 bp at the intronic regions flanking the 3′ splice site and overabundance of cis-regulatory elements, which are characteristic of alternatively spliced tandem acceptor sites and can distinguish them from the constitutive sites in which the proximal NAG splice site is selected. Our findings imply that AS at tandem splice sites and constitutive splicing of the distal NAG are highly regulated.     

Using estimative reaction free energy to predict splice sites and their flanking competitors

A crucial part in the gene structure prediction is to identify the accurate splice sites, not only constitutive but also alternative ones. Here, we use the maximum information principle (MIP) to analyze the conservative segments around splice sites. According to the MIP, a reaction free energy (RFE) expression is deduced, which can be employed to estimate the free energy change during splicing reaction involving a donor or acceptor site. The expression contains not only the background probability factors, but also all kinds of dependencies among both adjacent and non-adjacent bases. We apply the RFE expression to recognize splice sites and their flanking competitors in human genes, the results show high sensitivity and specificity, so the RFE expression accords well with the splicing reaction process. Moreover, the RFE expression is better than previous methods for predicting competitors of splice sites, and it outperforms the reaction free energy subtraction (RFES), that implies RFE competition between a given splice site and its flanking competitor may not be an only primary factor for alternative splice site selection. The work is helpful to not only the understanding of splicing reaction from its relation to MIP, but also the research on computational recognition of splicing sites and alternative splice events.     

Deep intron elements mediate nested splicing events at consecutive AG dinucleotides to regulate alternative 3' splice site choice in vertebrate 4.1 genes

Distal intraexon (iE) regulatory elements in 4.1R pre-mRNA govern 3' splice site choice at exon 2 (E2) via nested splicing events, ultimately modulating expression of N-terminal isoforms of cytoskeletal 4.1R protein. Here we explored intrasplicing in other normal and disease gene contexts and found conservation of intrasplicing through vertebrate evolution. In the paralogous 4.1B gene, we identified ～120 kb upstream of E2 an ultradistal intraexon, iE(B), that mediates intrasplicing by promoting two intricately coupled splicing events that ensure selection of a weak distal acceptor at E2 (E2dis) by prior excision of the competing proximal acceptor (E2prox). Mutating iE(B) in minigene splicing reporters abrogated intrasplicing, as did blocking endogenous iE(B) function with antisense morpholinos in live mouse and zebrafish animal models. In a human elliptocytosis patient with a mutant 4.1R gene lacking E2 through E4, we showed that aberrant splicing is consistent with iE(R)-mediated intrasplicing at the first available exons downstream of iE(R), namely, alternative E5 and constitutive E6. Finally, analysis of heterologous acceptor contexts revealed a strong preference for nested 3' splice events at consecutive pairs of AG dinucleotides. Distal regulatory elements may control intrasplicing at a subset of alternative 3' splice sites in vertebrate pre-mRNAs to generate proteins with functional diversity.     

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.     

Analysis of splice donor and acceptor site function in a transposable gene trap derived from the maize element Activator

Gene trap vectors have been used in insertional mutagenesis in animal systems to clone genes with interesting patterns of expression. These vectors are designed to allow the expression of a reporter gene when the vector inserts into a transcribed region. In this paper we examine alternative splicing events that result in the expression of a GUS reporter gene carried on a Ds element which has been designed as a gene trap vector for plants. We have developed a rapid and reliable method based on PCR to study such events. Many splice donor sites were observed in the 3′ Ac border. The relative frequency of utilisation of certain splice donor and acceptor sites differed between tobacco and Arabidopsis. A higher stringency of splicing was observed in Arabidopsis.     

Evidence for widespread association of mammalian splicing and conserved long-range RNA structures

Pre-mRNA structure impacts many cellular processes, including splicing in genes associated with disease. The contemporary paradigm of RNA structure prediction is biased toward secondary structures that occur within short ranges of pre-mRNA, although long-range base-pairings are known to be at least as important. Recently, we developed an efficient method for detecting conserved RNA structures on the genome-wide scale, one that does not require multiple sequence alignments and works equally well for the detection of local and long-range base-pairings. Using an enhanced method that detects base-pairings at all possible combinations of splice sites within each gene, we now report RNA structures that could be involved in the regulation of splicing in mammals. Statistically, we demonstrate strong association between the occurrence of conserved RNA structures and alternative splicing, where local RNA structures are generally more frequent at alternative donor splice sites, while long-range structures are more associated with weak alternative acceptor splice sites. As an example, we validated the RNA structure in the human SF1 gene using minigenes in the HEK293 cell line. Point mutations that disrupted the base-pairing of two complementary boxes between exons 9 and 10 of this gene altered the splicing pattern, while the compensatory mutations that reestablished the base-pairing reverted splicing to that of the wild-type. There is statistical evidence for a Dscam-like class of mammalian genes, in which mutually exclusive RNA structures control mutually exclusive alternative splicing. In sum, we propose that long-range base-pairings carry an important, yet unconsidered part of the splicing code, and that, even by modest estimates, there must be thousands of such potentially regulatory structures conserved throughout the evolutionary history of mammals.     

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。     

Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branchpoints for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism by which alternative promoters can be coordinated with downstream alternative splicing.