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      Spliced Leader Trapping Reveals Widespread Alternative Splicing Patterns in the Highly Dynamic Transcriptome of Trypanosoma brucei

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5′ splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

          Author Summary

          Some organisms like the human and animal parasite Trypanosoma brucei add a leader sequence to their mRNAs through a reaction called trans-splicing. Until now the splice sites for most mRNAs were unknown in T. brucei. Using high throughput sequencing we have developed a method to identify the splice sites and at the same time measure the abundance of the corresponding mRNAs. Analyzing three different life cycle stages of the parasite we identified the vast majority of splice sites in the organism and, to our great surprise, uncovered more than 2500 alternative splicing events, many of which appeared to be specific for one of the life cycle stages. Alternative splicing is a result of the addition of the leader sequence to different positions on the mRNA, leading to mixed mRNA populations that can encode for proteins with varying properties. One of the most obvious changes caused by alternative splicing is the gain or loss of targeting signals, leading to differential localization of the corresponding proteins. Based on our findings we hypothesize that alternative splicing is a major mechanism to regulate gene expression in T. brucei and could contribute to protein diversity in the parasite.

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          Most cited references 57

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          KEGG: kyoto encyclopedia of genes and genomes.

           S. Goto,  M Kanehisa (2000)
          KEGG (Kyoto Encyclopedia of Genes and Genomes) is a knowledge base for systematic analysis of gene functions, linking genomic information with higher order functional information. The genomic information is stored in the GENES database, which is a collection of gene catalogs for all the completely sequenced genomes and some partial genomes with up-to-date annotation of gene functions. The higher order functional information is stored in the PATHWAY database, which contains graphical representations of cellular processes, such as metabolism, membrane transport, signal transduction and cell cycle. The PATHWAY database is supplemented by a set of ortholog group tables for the information about conserved subpathways (pathway motifs), which are often encoded by positionally coupled genes on the chromosome and which are especially useful in predicting gene functions. A third database in KEGG is LIGAND for the information about chemical compounds, enzyme molecules and enzymatic reactions. KEGG provides Java graphics tools for browsing genome maps, comparing two genome maps and manipulating expression maps, as well as computational tools for sequence comparison, graph comparison and path computation. The KEGG databases are daily updated and made freely available (http://www. genome.ad.jp/kegg/).
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            WebLogo: a sequence logo generator.

            WebLogo generates sequence logos, graphical representations of the patterns within a multiple sequence alignment. Sequence logos provide a richer and more precise description of sequence similarity than consensus sequences and can rapidly reveal significant features of the alignment otherwise difficult to perceive. Each logo consists of stacks of letters, one stack for each position in the sequence. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of symbols within the stack reflects the relative frequency of the corresponding amino or nucleic acid at that position. WebLogo has been enhanced recently with additional features and options, to provide a convenient and highly configurable sequence logo generator. A command line interface and the complete, open WebLogo source code are available for local installation and customization. Copyright 2004 Cold Spring Harbor Laboratory Press
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              The significance of digital gene expression profiles.

              Genes differentially expressed in different tissues, during development, or during specific pathologies are of foremost interest to both basic and pharmaceutical research. "Transcript profiles" or "digital Northerns" are generated routinely by partially sequencing thousands of randomly selected clones from relevant cDNA libraries. Differentially expressed genes can then be detected from variations in the counts of their cognate sequence tags. Here we present the first systematic study on the influence of random fluctuations and sampling size on the reliability of this kind of data. We establish a rigorous significance test and demonstrate its use on publicly available transcript profiles. The theory links the threshold of selection of putatively regulated genes (e.g., the number of pharmaceutical leads) to the fraction of false positive clones one is willing to risk. Our results delineate more precisely and extend the limits within which digital Northern data can be used.
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                Author and article information

                Affiliations
                [1 ]Institute of Cell Biology, University of Bern, Bern, Switzerland
                [2 ]Fasteris, Genome Analyzer Service FASTERIS SA, Geneva, Switzerland
                Seattle Biomedical Research Institute, United States of America
                Author notes

                Conceived and designed the experiments: IR TO. Performed the experiments: DN KG MO TO. Analyzed the data: DN KG LB IR TO. Contributed reagents/materials/analysis tools: JM LF TO. Wrote the paper: IR TO.

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                August 2010
                August 2010
                5 August 2010
                : 6
                : 8
                2916883
                20700444
                09-PLPA-RA-2310R3
                10.1371/journal.ppat.1001037
                (Editor)
                Nilsson 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.
                Counts
                Pages: 13
                Categories
                Research Article
                Computational Biology/Alternative Splicing
                Infectious Diseases/Protozoal Infections
                Microbiology/Parasitology
                Molecular Biology/Bioinformatics
                Molecular Biology/mRNA Stability
                Molecular Biology/RNA Splicing

                Infectious disease & Microbiology

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