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Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm
RNA binding proteins (RBPs) regulate the lives of all RNAs from transcription, processing, and function to decay. How RNA–protein interactions change over time and space to support these roles is poorly understood. Towards this end, we sought to determine how two SR proteins—SRSF3 and SRSF7, regulat...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737842/ https://www.ncbi.nlm.nih.gov/pubmed/28977534 http://dx.doi.org/10.1093/nar/gkx671 |
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author | Brugiolo, Mattia Botti, Valentina Liu, Na Müller-McNicoll, Michaela Neugebauer, Karla M. |
author_facet | Brugiolo, Mattia Botti, Valentina Liu, Na Müller-McNicoll, Michaela Neugebauer, Karla M. |
author_sort | Brugiolo, Mattia |
collection | PubMed |
description | RNA binding proteins (RBPs) regulate the lives of all RNAs from transcription, processing, and function to decay. How RNA–protein interactions change over time and space to support these roles is poorly understood. Towards this end, we sought to determine how two SR proteins—SRSF3 and SRSF7, regulators of pre-mRNA splicing, nuclear export and translation—interact with RNA in different cellular compartments. To do so, we developed Fractionation iCLIP (Fr-iCLIP), in which chromatin, nucleoplasmic and cytoplasmic fractions are prepared from UV-crosslinked cells and then subjected to iCLIP. As expected, SRSF3 and SRSF7 targets were detected in all fractions, with intron, snoRNA and lncRNA interactions enriched in the nucleus. Cytoplasmically-bound mRNAs reflected distinct functional groupings, suggesting coordinated translation regulation. Surprisingly, hundreds of cytoplasmic intron targets were detected. These cytoplasmic introns were found to be highly conserved and introduced premature termination codons into coding regions. However, many intron-retained mRNAs were not substrates for nonsense-mediated decay (NMD), even though they were detected in polysomes. These findings suggest that intron-retained mRNAs in the cytoplasm have previously uncharacterized functions and/or escape surveillance. Hence, Fr-iCLIP detects the cellular location of RNA–protein interactions and provides insight into co-transcriptional, post-transcriptional and cytoplasmic RBP functions for coding and non-coding RNAs. |
format | Online Article Text |
id | pubmed-5737842 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-57378422018-01-04 Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm Brugiolo, Mattia Botti, Valentina Liu, Na Müller-McNicoll, Michaela Neugebauer, Karla M. Nucleic Acids Res Gene regulation, Chromatin and Epigenetics RNA binding proteins (RBPs) regulate the lives of all RNAs from transcription, processing, and function to decay. How RNA–protein interactions change over time and space to support these roles is poorly understood. Towards this end, we sought to determine how two SR proteins—SRSF3 and SRSF7, regulators of pre-mRNA splicing, nuclear export and translation—interact with RNA in different cellular compartments. To do so, we developed Fractionation iCLIP (Fr-iCLIP), in which chromatin, nucleoplasmic and cytoplasmic fractions are prepared from UV-crosslinked cells and then subjected to iCLIP. As expected, SRSF3 and SRSF7 targets were detected in all fractions, with intron, snoRNA and lncRNA interactions enriched in the nucleus. Cytoplasmically-bound mRNAs reflected distinct functional groupings, suggesting coordinated translation regulation. Surprisingly, hundreds of cytoplasmic intron targets were detected. These cytoplasmic introns were found to be highly conserved and introduced premature termination codons into coding regions. However, many intron-retained mRNAs were not substrates for nonsense-mediated decay (NMD), even though they were detected in polysomes. These findings suggest that intron-retained mRNAs in the cytoplasm have previously uncharacterized functions and/or escape surveillance. Hence, Fr-iCLIP detects the cellular location of RNA–protein interactions and provides insight into co-transcriptional, post-transcriptional and cytoplasmic RBP functions for coding and non-coding RNAs. Oxford University Press 2017-10-13 2017-08-10 /pmc/articles/PMC5737842/ /pubmed/28977534 http://dx.doi.org/10.1093/nar/gkx671 Text en © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Gene regulation, Chromatin and Epigenetics Brugiolo, Mattia Botti, Valentina Liu, Na Müller-McNicoll, Michaela Neugebauer, Karla M. Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm |
title | Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm |
title_full | Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm |
title_fullStr | Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm |
title_full_unstemmed | Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm |
title_short | Fractionation iCLIP detects persistent SR protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm |
title_sort | fractionation iclip detects persistent sr protein binding to conserved, retained introns in chromatin, nucleoplasm and cytoplasm |
topic | Gene regulation, Chromatin and Epigenetics |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737842/ https://www.ncbi.nlm.nih.gov/pubmed/28977534 http://dx.doi.org/10.1093/nar/gkx671 |
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