Cargando…
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...
Autores principales: | , |
---|---|
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 |
_version_ | 1782185524010680320 |
---|---|
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. |
format | Text |
id | pubmed-2923612 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2010 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT houseleyjonathan apparentnoncanonicaltranssplicingisgeneratedbyreversetranscriptaseinvitro AT tollerveydavid apparentnoncanonicaltranssplicingisgeneratedbyreversetranscriptaseinvitro |