Computational prediction of splicing regulatory elements shared by Tetrapoda organisms

Background: auxiliary splicing sequences play an important role in ensuring accurate and efficient splicing by promoting or repressing recognition of authentic splice sites. These cis-acting motifs have been termed splicing enhancers and silencers and are located both in introns and exons. They co-evolved into an intricate splicing code together with additional functional constraints, such as tissue-specific and alternative splicing patterns. We used orthologous exons extracted from the University of California Santa Cruz multiple genome alignments of human and 22 Tetrapoda organisms to predict candidate enhancers and silencers that have reproducible and statistically significant bias towards annotated exonic boundaries.Results: a total of 2,546 Tetrapoda enhancers and silencers were clustered into 15 putative core motifs based on their Markov properties. Most of these elements have been identified previously, but 118 putative silencers and 260 enhancers (~15%) were novel. Examination of previously published experimental data for the presence of predicted elements showed that their mutations in 21/23 (91.3%) cases altered the splicing pattern as expected. Predicted intronic motifs flanking 3' and 5' splice sites had higher evolutionary conservation than other sequences within intronic flanks and the intronic enhancers were markedly differed between 3' and 5' intronic flanks.Conclusion: difference in intronic enhancers supporting 5' and 3' splice sites suggests an independent splicing commitment for neighboring exons. Increased evolutionary conservation for ISEs/ISSs within intronic flanks and effect of modulation of predicted elements on splicing suggest functional significance of found elements in splicing regulation. Most of the elements identified were shown to have direct implications in human splicing and therefore could be useful for building computational splicing models in biomedical researc

Branch site haplotypes that control alternative splicing

We show that the allele-dependent expression of transcripts encoding soluble HLA-DQβ chains is determined by branchpoint sequence (BPS) haplotypes in DQB1 intron 3. BPS RNAs associated with low inclusion of the transmembrane exon in mature transcripts showed impaired binding to splicing factor 1 (SF1), indicating that alternative splicing of DQB1 is controlled by differential BPS recognition early during spliceosome assembly. We also demonstrate that naturally occurring human BPS point mutations that alter splicing and lead to recognizable phenotypes cluster in BP and in position −2 relative to BP, implicating impaired SF1-BPS interactions in disease-associated BPS substitutions. Coding DNA variants produced smaller fluctuations of exon inclusion levels than random exonic substitutions, consistent with a selection against coding mutations that alter their own exonization. Finally, proximal splicing in this multi-allelic reporter system was promoted by at least seven SR proteins and repressed by hnRNPs F, H and I, supporting an extensive antagonism of factors balancing the splice site selection. These results provide the molecular basis for the haplotype-specific expression of soluble DQβ, improve prediction of intronic point mutations and indicate how extraordinary, selection-driven DNA variability in HLA affects pre-mRNA splicin

Selection of olduvai domains during evolution: A role for primate-specific splicing super-enhancer and RNA Guanine Quadruplex in Bipartite NBPF Exons

Olduvai protein domains (also known as DUF1220 or NBPF) have undergone the greatest human-specific increase in the copy number of any coding region in the genome. Their repeat number was strongly associated with the evolutionary expansion of brain volumes, neuron counts and cognitive abilities, as well as with disorders of the autistic spectrum. Nevertheless, the domain function and cellular mechanisms underlying the positive selection of Olduvai DNA sequences in higher primates remain obscure. Here, I show that the inclusion of Olduvai exon doublets in mature transcripts is facilitated by a potent splicing enhancer that was created through duplication within the first exon. The enhancer is the strongest among the NBPF transcripts and further promotes the already high splicing activity of the unexpanded first exons of the two-exon domains, safeguarding the expanded Olduvai exon doublets in the mature transcriptome. The duplication also creates a predicted RNA guanine quadruplex that may regulate the access to spliceosomal components of the super-enhancer and influence the splicing of adjacent exons. Thus, positive Olduvai selection during primate evolution is likely to result from a combination of multiple targets in gene expression pathways, including RNA splicing.</p

Intergenic transcripts in genes with phase I introns

Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality-control mechanism that detects and degrades aberrant transcripts prematurely terminating translation. NMD may be elicited by intergenic transcripts that contain premature termination codons (PTCs), but chimeric mRNAs of genes that have introns of identical phase would be predicted to lack PTCs and escape NMD. We examined intron phase I-containing HLA class II genes for the presence of intergenic mRNAs and found an extraordinary diversity of correctly spliced and polyadenylated intergenic transcripts. They lacked a significant homology at the chimeric joins and had no PTCs. Their expression levels were very low and positively correlated with the expression of natural transcripts. In contrast, pair-wise mixtures of separately transcribed plasmids carrying full-length HLA-DQB1, -DQA1, -DRB1, and -DRA cDNAs produced only hybrid molecules that lacked canonical exon boundaries, had homologous chimeric joins, and occasionally contained PTCs, implicating in vitro artifacts generated by template switching of Taq polymerase and reverse transcriptase. A differential exon structure of hybrid molecules observed in vitro and in cellular RNA preparations suggests that intergenic mRNAs with canonical exon boundaries arise in vivo during exon joining and/or transcription. Since the observed intergenic mRNAs may encode mixed class II heterodimers that were previously shown to present antigens it will be interesting to determine functional properties of such molecules in future studies

Alu RNA fold links splicing with signal recognition particle proteins

Transcriptomic diversity in primates was considerably expanded by exonizations of intronic Alu elements. To better understand their cellular mechanisms we have used structure-based mutagenesis coupled with functional and proteomic assays to study the impact of successive primate mutations and their combinations on inclusion of a sense-oriented AluJ exon in the human F8 gene. We show that the splicing outcome was better predicted by consecutive RNA conformation changes than by computationally derived splicing regulatory motifs. We also demonstrate an involvement of SRP9/14 (signal recognition particle) heterodimer in splicing regulation of Alu-derived exons. Nucleotide substitutions that accumulated during primate evolution relaxed the conserved left-arm AluJ structure including helix H1 and reduced the capacity of SRP9/14 to stabilize the closed Alu conformation. RNA secondary structure-constrained mutations that promoted open Y-shaped conformations of the Alu made the Alu exon inclusion reliant on DHX9. Finally, we identified additional SRP9/14 sensitive Alu exons and predicted their functional roles in the cell. Together, these results provide unique insights into architectural elements required for sense Alu exonization, identify conserved pre-mRNA structures involved in exon selection and point to a possible chaperone activity of SRP9/14 outside the mammalian signal recognition particle

Exonization of AluYa5 in the human ACE gene requires mutations in both 3' and 5' splice sites and is facilitated by a conserved splicing enhancer

Ancient Alu elements have been shown to be included in mature transcripts by point mutations that improve their 5' or 3' splice sites. We have examined requirements for exonization of a younger, disease-associated AluYa5 in intron 16 of the human ACE gene. A single G&gt;C transversion in position –3 of the new Alu exon was insufficient for Alu exonization and a significant inclusion in mRNA was only observed when improving several potential splice donor sites in the presence of 3' CAG. Since complete Alu exonization was not achieved by optimizing traditional splicing signals, including the branch site, we tested whether auxiliary elements in AluYa5 were required for constitutive inclusion. Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 222–228 and point mutations in highly conserved nucleotides of this heptamer decreased Alu inclusion. In addition, we show that Alu exonization was facilitated by a subset of serine/arginine-rich (SR) proteins through activation of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE minigenes generated similar splicing patterns in both ACE-expressing and non-expressing cells, suggesting that previously reported allelic association with plasma ACE activity and cardiovascular disease is not attributable to differential splicing of introns 16 and 17

A method of predicting changes in human gene splicing induced by genetic variants in context of cis-acting elements

Background: polymorphic variants and mutations disrupting canonical splicing isoforms are among the leading causes of human hereditary disorders. While there is a substantial evidence of aberrant splicing causing Mendelian diseases, the implication of such events in multi-genic disorders is yet to be well understood. We have developed a new tool (SpliceScan II) for predicting the effects of genetic variants on splicing and cis-regulatory elements. The novel Bayesian non-canonical 5’GC splice site (SS) sensor used in our tool allows inference on non-canonical exons. Result: our tool performed favorably when compared with the existing methods in the context of genes linked to the Autism Spectrum Disorder (ASD). SpliceScan II was able to predict more aberrant splicing isoforms triggered by the mutations, as documented in DBASS5 and DBASS3 aberrant splicing databases, than other existing methods. Detrimental effects behind some of the polymorphic variations previously associated with Alzheimer’s and breast cancer could be explained by changes in predicted splicing patterns. Conclusions: we have developed SpliceScan II, an effective and sensitive tool for predicting the detrimental effects of genomic variants on splicing leading to Mendelian and complex hereditary disorders. The method could potentially be used to screen resequenced patient DNA to identify de novo mutations and polymorphic variants that could contribute to a genetic disorde