Hett, A. and West, S. (2014) Inhibition of U4 snRNA in Human Cells Causes the Stable Retention of Polyadenylated Pre-mRNA in the Nucleus. PLoS ONE, 9 (5). ARTN e96174. ISSN 1932-6203
Abstract
Most human pre-mRNAs contain introns that are removed by splicing. Such a complex process needs strict control and regulation in order to prevent the expression of aberrant or unprocessed transcripts. To analyse the fate of pre-mRNAs that cannot be spliced, we inhibited splicing using an anti-sense morpholino (AMO) against U4 snRNA. As a consequence, splicing of several selected transcripts was strongly inhibited. This was accompanied by the formation of enlarged nuclear speckles containing polyadenylated RNA, splicing factors and the nuclear poly(A) binding protein. Consistently, more polyadenylated pre-mRNA could be isolated from nucleoplasmic as well as chromatin-associated RNA fractions following U4 inhibition. Further analysis demonstrated that accumulated pre-mRNAs were stable in the nucleus and that nuclear RNA degradation factors did not re-localise to nuclear speckles following splicing inhibition. The accumulation of pre-mRNA and the formation of enlarged speckles were sensitive to depletion of the 3′ end processing factor, CPSF73, suggesting a requirement for poly(A) site processing in this mechanism. Finally, we provide evidence that the pre-mRNAs produced following U4 snRNA inhibition remain competent for splicing, perhaps providing a biological explanation for their stability. These data further characterise processes ensuring the nuclear retention of pre-mRNA that cannot be spliced and suggest that, in some cases, unspliced transcripts can complete splicing sometime after their initial synthesis.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2014 Hett, West. 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. https://creativecommons.org/licenses/by/4.0/ |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) > Department of Molecular Biology and Biotechnology (Sheffield) |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 05 Dec 2016 15:52 |
Last Modified: | 05 Dec 2016 15:58 |
Published Version: | http://dx.doi.org/10.1371/journal.pone.0096174 |
Status: | Published |
Publisher: | Public Library of Science |
Refereed: | Yes |
Identification Number: | 10.1371/journal.pone.0096174 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:108673 |