A class of human exons with predicted distant branch points revealed by analysis of AG dinucleotide exclusion zones

Background: The three consensus elements at the 3' end of human introns-the branch point sequence, the polypyrimidine tract, and the 3' splice site AG dinucleotide-are usually closely spaced within the final 40 nucleotides of the intron. However, the branch point sequence and polypyrimidine tract of a few known alternatively spliced exons lie up to 400 nucleotides upstream of the 3' splice site. The extended regions between the distant branch points (dBPs) and their 3' splice site are marked by the absence of other AG dinucleotides. In many cases alternative splicing regulatory elements are located within this region.|Results: We have applied a simple algorithm, based on AG dinucleotide exclusion zones (AGEZ), to a large data set of verified human exons. We found a substantial number of exons with large AGEZs, which represent candidate dBP exons. We verified the importance of the predicted dBPs for splicing of some of these exons. This group of exons exhibits a higher than average prevalence of observed alternative splicing, and many of the exons are in genes with some human disease association.|Conclusion: The group of identified probable dBP exons are interesting first because they are likely to be alternatively spliced. Second, they are expected to be vulnerable to mutations within the entire extended AGEZ. Disruption of splicing of such exons, for example by mutations that lead to insertion of a new AG dinucleotide between the dBP and 3' splice site, could be readily understood even though the causative mutation might be remote from the conventional locations of splice site sequences.This work was funded by programme grant 059879 from the Wellcome Trust to C.W.J.S

An apparent pseudo-exon acts both as an alternative exon that leads to nonsense-mediated decay and as a zero-length exon.

Pseudo-exons are intronic sequences that are flanked by apparent consensus splice sites but that are not observed in spliced mRNAs. Pseudo-exons are often difficult to activate by mutation and have typically been viewed as a conceptual challenge to our understanding of how the spliceosome discriminates between authentic and cryptic splice sites. We have analyzed an apparent pseudo-exon located downstream of mutually exclusive exons 2 and 3 of the rat α-tropomyosin (TM) gene. The TM pseudo-exon is conserved among mammals and has a conserved profile of predicted splicing enhancers and silencers that is more typical of a genuine exon than a pseudo-exon. Splicing of the pseudo-exon is fully activated for splicing to exon 3 by a number of simple mutations. Splicing of the pseudo-exon to exon 3 is predicted to lead to nonsense-mediated decay (NMD). In contrast, when “prespliced” to exon 2 it follows a “zero length exon” splicing pathway in which a newly generated 5′ splice site at the junction with exon 2 is spliced to exon 4. We propose that a subset of apparent pseudo-exons, as exemplified here, are actually authentic alternative exons whose inclusion leads to NMD

Molecular design of a splicing switch responsive to the RNA binding protein Tra2β

Tra2β regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase 3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2β most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2β were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2β AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2β-mediated activation, although single mutated exons could still bind Tra2β protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2β-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5′-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 5′-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2β, and co-ordinately regulates HIPK3-T exon splicing inclusion

Detecting translational regulation by change point analysis of ribosome profiling data sets

Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While during normal translation, ribosomedensity is constant along the length of the mRNA coding region, this can be altered in response to translational regulatoryevents. In the present study, we developed a method to detect translational regulation of individual mRNAs from their ribosomeprofiles, utilizing changes in ribosome density. We used mathematical modeling to show that changes in ribosome density shouldoccur along the mRNA at the point of regulation. We analyzed a Ribo-Seq data set obtained for mouse embryonic stem cells andshowed that normalization by corresponding RNA-Seq can be used to improve the Ribo-Seq quality by removing bias introducedby deep-sequencing and alignment artifacts. After normalization, we applied a change point algorithm to detect changes inribosome density present in individual mRNA ribosome profiles. Additional sequence and gene isoform information obtained fromthe UCSC Genome Browser allowed us to further categorize the detected changes into different mechanisms of regulation. Inparticular, we detected several mRNAs with known post-transcriptional regulation, e.g., premature termination for selenoproteinmRNAs and translational control of Atf4, but also several more mRNAs with hitherto unknown translational regulation. Additionally, our approach proved useful foridentification of new transcript isoforms