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Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro

BACKGROUND: Trans-splicing, the in vivo joining of two independently transcribed RNA molecules, is well characterized in lower eukaryotes, but was long thought absent from metazoans. However, recent bioinformatic analyses of EST sequences suggested widespread trans-splicing in mammals. These apparen...

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Detalles Bibliográficos
Autores principales: Houseley, Jonathan, Tollervey, David
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923612/
https://www.ncbi.nlm.nih.gov/pubmed/20805885
http://dx.doi.org/10.1371/journal.pone.0012271
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author Houseley, Jonathan
Tollervey, David
author_facet Houseley, Jonathan
Tollervey, David
author_sort Houseley, Jonathan
collection PubMed
description BACKGROUND: Trans-splicing, the in vivo joining of two independently transcribed RNA molecules, is well characterized in lower eukaryotes, but was long thought absent from metazoans. However, recent bioinformatic analyses of EST sequences suggested widespread trans-splicing in mammals. These apparently spliced transcripts generally lacked canonical splice sites, leading us to question their authenticity. Particularly, the native ability of reverse transcriptase enzymes to template switch during transcription could produce apparently trans-spliced sequences. PRINCIPAL FINDINGS: Here we report an in vitro system for the analysis of template switching in reverse transcription. Using highly purified RNA substrates, we show the reproducible occurrence of apparent trans-splicing between two RNA molecules. Other reported non-canonical splicing events such as exon shuffling and sense-antisense fusions were also readily detected. The latter caused the production of apparent antisense non-coding RNAs, which are also reported to be abundant in humans. CONCLUSIONS: We propose that most reported examples of non-canonical splicing in metazoans arise through template switching by reverse transcriptase during cDNA preparation. We further show that the products of template switching can vary between reverse transcriptases, providing a simple diagnostic for identifying many of these experimental artifacts.
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spelling pubmed-29236122010-08-30 Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro Houseley, Jonathan Tollervey, David PLoS One Research Article BACKGROUND: Trans-splicing, the in vivo joining of two independently transcribed RNA molecules, is well characterized in lower eukaryotes, but was long thought absent from metazoans. However, recent bioinformatic analyses of EST sequences suggested widespread trans-splicing in mammals. These apparently spliced transcripts generally lacked canonical splice sites, leading us to question their authenticity. Particularly, the native ability of reverse transcriptase enzymes to template switch during transcription could produce apparently trans-spliced sequences. PRINCIPAL FINDINGS: Here we report an in vitro system for the analysis of template switching in reverse transcription. Using highly purified RNA substrates, we show the reproducible occurrence of apparent trans-splicing between two RNA molecules. Other reported non-canonical splicing events such as exon shuffling and sense-antisense fusions were also readily detected. The latter caused the production of apparent antisense non-coding RNAs, which are also reported to be abundant in humans. CONCLUSIONS: We propose that most reported examples of non-canonical splicing in metazoans arise through template switching by reverse transcriptase during cDNA preparation. We further show that the products of template switching can vary between reverse transcriptases, providing a simple diagnostic for identifying many of these experimental artifacts. Public Library of Science 2010-08-18 /pmc/articles/PMC2923612/ /pubmed/20805885 http://dx.doi.org/10.1371/journal.pone.0012271 Text en Houseley, Tollervey. http://creativecommons.org/licenses/by/4.0/ 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 properly credited.
spellingShingle Research Article
Houseley, Jonathan
Tollervey, David
Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro
title Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro
title_full Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro
title_fullStr Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro
title_full_unstemmed Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro
title_short Apparent Non-Canonical Trans-Splicing Is Generated by Reverse Transcriptase In Vitro
title_sort apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923612/
https://www.ncbi.nlm.nih.gov/pubmed/20805885
http://dx.doi.org/10.1371/journal.pone.0012271
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