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Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays

Most human pathogenic mutations in 5′ splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in ∼1% of all human 5′ss. It is therefore a challenging task to understa...

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Autores principales: Erkelenz, Steffen, Theiss, Stephan, Kaisers, Wolfgang, Ptok, Johannes, Walotka, Lara, Müller, Lisa, Hillebrand, Frank, Brillen, Anna-Lena, Sladek, Michael, Schaal, Heiner
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280755/
https://www.ncbi.nlm.nih.gov/pubmed/30355602
http://dx.doi.org/10.1101/gr.235861.118
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author Erkelenz, Steffen
Theiss, Stephan
Kaisers, Wolfgang
Ptok, Johannes
Walotka, Lara
Müller, Lisa
Hillebrand, Frank
Brillen, Anna-Lena
Sladek, Michael
Schaal, Heiner
author_facet Erkelenz, Steffen
Theiss, Stephan
Kaisers, Wolfgang
Ptok, Johannes
Walotka, Lara
Müller, Lisa
Hillebrand, Frank
Brillen, Anna-Lena
Sladek, Michael
Schaal, Heiner
author_sort Erkelenz, Steffen
collection PubMed
description Most human pathogenic mutations in 5′ splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in ∼1% of all human 5′ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5′ss are used. In this work, we systematically examined noncanonical 5′ splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5′ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of U1 snRNA complementarity in the competing 5′ss. In particular, we could confirm splicing at different positions (i.e., −1, +1, +5) of a splice site for all noncanonical dinucleotides “weaker” than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5′ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5′ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking.
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spelling pubmed-62807552019-06-01 Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays Erkelenz, Steffen Theiss, Stephan Kaisers, Wolfgang Ptok, Johannes Walotka, Lara Müller, Lisa Hillebrand, Frank Brillen, Anna-Lena Sladek, Michael Schaal, Heiner Genome Res Research Most human pathogenic mutations in 5′ splice sites affect the canonical GT in positions +1 and +2, leading to noncanonical dinucleotides. On the other hand, noncanonical dinucleotides are observed under physiological conditions in ∼1% of all human 5′ss. It is therefore a challenging task to understand the pathogenic mutation mechanisms underlying the conditions under which noncanonical 5′ss are used. In this work, we systematically examined noncanonical 5′ splice site selection, both experimentally using splicing competition reporters and by analyzing a large RNA-seq data set of 54 fibroblast samples from 27 subjects containing a total of 2.4 billion gapped reads covering 269,375 exon junctions. From both approaches, we consistently derived a noncanonical 5′ss usage ranking GC > TT > AT > GA > GG > CT. In our competition splicing reporter assay, noncanonical splicing was strictly dependent on the presence of upstream or downstream splicing regulatory elements (SREs), and changes in SREs could be compensated by variation of U1 snRNA complementarity in the competing 5′ss. In particular, we could confirm splicing at different positions (i.e., −1, +1, +5) of a splice site for all noncanonical dinucleotides “weaker” than GC. In our comprehensive RNA-seq data set analysis, noncanonical 5′ss were preferentially detected in weakly used exon junctions of highly expressed genes. Among high-confidence splice sites, they were 10-fold overrepresented in clusters with a neighboring, more frequently used 5′ss. Conversely, these more frequently used neighbors contained only the dinucleotides GT, GC, and TT, in accordance with the above ranking. Cold Spring Harbor Laboratory Press 2018-12 /pmc/articles/PMC6280755/ /pubmed/30355602 http://dx.doi.org/10.1101/gr.235861.118 Text en © 2018 Erkelenz et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Research
Erkelenz, Steffen
Theiss, Stephan
Kaisers, Wolfgang
Ptok, Johannes
Walotka, Lara
Müller, Lisa
Hillebrand, Frank
Brillen, Anna-Lena
Sladek, Michael
Schaal, Heiner
Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays
title Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays
title_full Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays
title_fullStr Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays
title_full_unstemmed Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays
title_short Ranking noncanonical 5′ splice site usage by genome-wide RNA-seq analysis and splicing reporter assays
title_sort ranking noncanonical 5′ splice site usage by genome-wide rna-seq analysis and splicing reporter assays
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280755/
https://www.ncbi.nlm.nih.gov/pubmed/30355602
http://dx.doi.org/10.1101/gr.235861.118
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