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Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex

SF3b1 is an essential component of the U2 snRNP implicated in branch site (BS) recognition and found to be frequently mutated in several human cancers. While recent structures of yeast and human SF3b1 have revealed its molecular architecture, the importance of specific RNA:protein contacts and confo...

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Autores principales: Carrocci, Tucker J., Paulson, Joshua C., Hoskins, Aaron A.
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/PMC6049509/
https://www.ncbi.nlm.nih.gov/pubmed/29752352
http://dx.doi.org/10.1261/rna.065664.118
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author Carrocci, Tucker J.
Paulson, Joshua C.
Hoskins, Aaron A.
author_facet Carrocci, Tucker J.
Paulson, Joshua C.
Hoskins, Aaron A.
author_sort Carrocci, Tucker J.
collection PubMed
description SF3b1 is an essential component of the U2 snRNP implicated in branch site (BS) recognition and found to be frequently mutated in several human cancers. While recent structures of yeast and human SF3b1 have revealed its molecular architecture, the importance of specific RNA:protein contacts and conformational changes remains largely uncharacterized. Here, we performed mutational analysis of yeast SF3b1, guided by recent structures of the spliceosome. We find that conserved amino acids contacting the U2 snRNA backbone of the U2/BS duplex are nonessential, and that yeast can tolerate truncation of the HEAT repeats containing these amino acids. The pocket housing the branchpoint adenosine (BP-A) is also amenable to mutation despite strong conservation. However, mutations that support viability can still lead to defects in splicing pre-mRNAs with nonconsensus BS substitutions found at −3, −2, −1, and +1 positions relative to the BP-A or at the branchpoint position. Through the generation of yeast and human chimeric proteins, we further defined the functionally conserved regions of Hsh155 as well as identify changes in BS usage resulting from inclusion of human SF3b1 HEAT repeats. Moreover, these chimeric proteins confer a sensitivity to small molecule inhibition by pladienolide B to yeast splicing. Together, these data reveal the importance of individual contacts of Hsh155/SF3b1 to the U2/BS duplex and define their contribution to BS usage by the spliceosome.
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spelling pubmed-60495092019-08-01 Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex Carrocci, Tucker J. Paulson, Joshua C. Hoskins, Aaron A. RNA Article SF3b1 is an essential component of the U2 snRNP implicated in branch site (BS) recognition and found to be frequently mutated in several human cancers. While recent structures of yeast and human SF3b1 have revealed its molecular architecture, the importance of specific RNA:protein contacts and conformational changes remains largely uncharacterized. Here, we performed mutational analysis of yeast SF3b1, guided by recent structures of the spliceosome. We find that conserved amino acids contacting the U2 snRNA backbone of the U2/BS duplex are nonessential, and that yeast can tolerate truncation of the HEAT repeats containing these amino acids. The pocket housing the branchpoint adenosine (BP-A) is also amenable to mutation despite strong conservation. However, mutations that support viability can still lead to defects in splicing pre-mRNAs with nonconsensus BS substitutions found at −3, −2, −1, and +1 positions relative to the BP-A or at the branchpoint position. Through the generation of yeast and human chimeric proteins, we further defined the functionally conserved regions of Hsh155 as well as identify changes in BS usage resulting from inclusion of human SF3b1 HEAT repeats. Moreover, these chimeric proteins confer a sensitivity to small molecule inhibition by pladienolide B to yeast splicing. Together, these data reveal the importance of individual contacts of Hsh155/SF3b1 to the U2/BS duplex and define their contribution to BS usage by the spliceosome. Cold Spring Harbor Laboratory Press 2018-08 /pmc/articles/PMC6049509/ /pubmed/29752352 http://dx.doi.org/10.1261/rna.065664.118 Text en © 2018 Carrocci et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 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 Article
Carrocci, Tucker J.
Paulson, Joshua C.
Hoskins, Aaron A.
Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex
title Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex
title_full Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex
title_fullStr Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex
title_full_unstemmed Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex
title_short Functional analysis of Hsh155/SF3b1 interactions with the U2 snRNA/branch site duplex
title_sort functional analysis of hsh155/sf3b1 interactions with the u2 snrna/branch site duplex
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6049509/
https://www.ncbi.nlm.nih.gov/pubmed/29752352
http://dx.doi.org/10.1261/rna.065664.118
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