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      Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution

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          Abstract

          Inclusion or exclusion of single codons at the splice acceptor site of mammalian genes is regulated in a tissue-specific manner, is strongly conserved, and is associated with local accelerated protein evolution.

          Abstract

          Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3′ splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ∼15-fold increase in the frequency of three base pair gaps at 3′ splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.

          Author Summary

          In order to translate a gene into protein, all of the non-coding regions (introns) need to be removed from the transcript and the coding regions (exons) stitched back together to make an mRNA. Most human genes are alternatively spliced, allowing the selection of different combinations of exons to produce multiple distinct mRNAs and proteins. Many types of alternative splicing are known to play crucial roles in biological processes including cell fate determination, tumor metabolism, and apoptosis. In this study, we investigated a form of alternative splicing in which competing adjacent 3′ splice sites (or splice acceptor sites) generate mRNAs differing by just an RNA triplet, the size of a single codon. This mode of alternative splicing, known as NAGNAG splicing, affects thousands of human genes and has been known for a decade, but its potential regulation, physiological importance, and conservation across species have been disputed. Using high-throughput sequencing of cDNA (“RNA-Seq”) from human and mouse tissues, we found that single-codon splicing often shows strong tissue specificity. Regulated NAGNAG alternative splice sites are selectively conserved between human and mouse genes, suggesting that they are important for organismal fitness. We identified features of the competing splice sites that influence NAGNAG splicing, and validated their effects in cultured cells. Furthermore, we found that this mode of splicing is associated with accelerated and highly biased protein evolution at exon boundaries. Taken together, our analyses demonstrate that the inclusion or exclusion of RNA triplets at exon boundaries can be effectively regulated by the splicing machinery, and highlight an unexpected connection between RNA processing and protein evolution.

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          The Bioperl toolkit: Perl modules for the life sciences.

          The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort.
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            The UCSC Genome Browser database: update 2011

            The University of California, Santa Cruz Genome Browser (http://genome.ucsc.edu) offers online access to a database of genomic sequence and annotation data for a wide variety of organisms. The Browser also has many tools for visualizing, comparing and analyzing both publicly available and user-generated genomic data sets, aligning sequences and uploading user data. Among the features released this year are a gene search tool and annotation track drag-reorder functionality as well as support for BAM and BigWig/BigBed file formats. New display enhancements include overlay of multiple wiggle tracks through use of transparent coloring, options for displaying transformed wiggle data, a ‘mean+whiskers’ windowing function for display of wiggle data at high zoom levels, and more color schemes for microarray data. New data highlights include seven new genome assemblies, a Neandertal genome data portal, phenotype and disease association data, a human RNA editing track, and a zebrafish Conservation track. We also describe updates to existing tracks.
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              Understanding alternative splicing: towards a cellular code.

              In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms - thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches. These promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.
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                Author and article information

                Contributors
                Role: Academic Editor
                Journal
                PLoS Biol
                plos
                plosbiol
                PLoS Biology
                Public Library of Science (San Francisco, USA )
                1544-9173
                1545-7885
                January 2012
                January 2012
                3 January 2012
                : 10
                : 1
                : e1001229
                Affiliations
                [1]Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America
                University of Bath, United Kingdom
                Author notes

                The author(s) have made the following declarations about their contributions: Conceived and designed the experiments: RKB JM NJL CBB. Performed the experiments: RKB NL JM. Analyzed the data: RKB. Contributed reagents/materials/analysis tools: JM. Wrote the paper: RKB CBB.

                ¤: Current address: Computational Biology Program, Public Health Sciences Division, and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America

                Article
                PBIOLOGY-D-11-02569
                10.1371/journal.pbio.1001229
                3250501
                22235189
                4106c6bc-3761-46be-9bf4-9e4abffdf2aa
                Bradley et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 23 June 2011
                : 18 November 2011
                Page count
                Pages: 15
                Categories
                Research Article
                Biology
                Computational Biology
                Evolutionary Biology
                Genetics
                Genomics

                Life sciences
                Life sciences

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