Cargando…

The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing

Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin...

Descripción completa

Detalles Bibliográficos
Autores principales: Hadjivassiliou, Haralambos, Rosenberg, Oren S., Guthrie, Christine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988567/
https://www.ncbi.nlm.nih.gov/pubmed/24681967
http://dx.doi.org/10.1261/rna.042838.113
_version_ 1782312033807499264
author Hadjivassiliou, Haralambos
Rosenberg, Oren S.
Guthrie, Christine
author_facet Hadjivassiliou, Haralambos
Rosenberg, Oren S.
Guthrie, Christine
author_sort Hadjivassiliou, Haralambos
collection PubMed
description Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1's ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1's ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1's iUSP domain to interact with the spliceosome.
format Online
Article
Text
id pubmed-3988567
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Cold Spring Harbor Laboratory Press
record_format MEDLINE/PubMed
spelling pubmed-39885672015-05-01 The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing Hadjivassiliou, Haralambos Rosenberg, Oren S. Guthrie, Christine RNA Articles Sad1 is an essential splicing factor initially identified in a genetic screen in Saccharomyces cerevisiae for snRNP assembly defects. Based on sequence homology, Sad1, or USP39 in humans, is predicted to comprise two domains: a zinc finger ubiquitin binding domain (ZnF-UBP) and an inactive ubiquitin-specific protease (iUSP) domain, both of which are well conserved. The role of these domains in splicing and their interaction with ubiquitin are unknown. We first used splicing microarrays to analyze Sad1 function in vivo and found that Sad1 is critical for the splicing of nearly all yeast intron-containing genes. By using in vitro assays, we then showed that it is required for the assembly of the active spliceosome. To gain structural insights into Sad1 function, we determined the crystal structure of the full-length protein at 1.8 Å resolution. In the structure, the iUSP domain forms the characteristic ubiquitin binding pocket, though with an amino acid substitution in the active site that results in complete inactivation of the enzymatic activity of the domain. The ZnF-UBP domain of Sad1 shares high structural similarly to other ZnF-UBPs; however, Sad1's ZnF-UBP does not possess the canonical ubiquitin binding motif. Given the precedents for ZnF-UBP domains to function as activators for their neighboring USP domains, we propose that Sad1's ZnF-UBP acts in a ubiquitin-independent capacity to recruit and/or activate Sad1's iUSP domain to interact with the spliceosome. Cold Spring Harbor Laboratory Press 2014-05 /pmc/articles/PMC3988567/ /pubmed/24681967 http://dx.doi.org/10.1261/rna.042838.113 Text en © 2014 Hadjivassiliou 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 Articles
Hadjivassiliou, Haralambos
Rosenberg, Oren S.
Guthrie, Christine
The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing
title The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing
title_full The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing
title_fullStr The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing
title_full_unstemmed The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing
title_short The crystal structure of S. cerevisiae Sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mRNA splicing
title_sort crystal structure of s. cerevisiae sad1, a catalytically inactive deubiquitinase that is broadly required for pre-mrna splicing
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988567/
https://www.ncbi.nlm.nih.gov/pubmed/24681967
http://dx.doi.org/10.1261/rna.042838.113
work_keys_str_mv AT hadjivassiliouharalambos thecrystalstructureofscerevisiaesad1acatalyticallyinactivedeubiquitinasethatisbroadlyrequiredforpremrnasplicing
AT rosenbergorens thecrystalstructureofscerevisiaesad1acatalyticallyinactivedeubiquitinasethatisbroadlyrequiredforpremrnasplicing
AT guthriechristine thecrystalstructureofscerevisiaesad1acatalyticallyinactivedeubiquitinasethatisbroadlyrequiredforpremrnasplicing
AT hadjivassiliouharalambos crystalstructureofscerevisiaesad1acatalyticallyinactivedeubiquitinasethatisbroadlyrequiredforpremrnasplicing
AT rosenbergorens crystalstructureofscerevisiaesad1acatalyticallyinactivedeubiquitinasethatisbroadlyrequiredforpremrnasplicing
AT guthriechristine crystalstructureofscerevisiaesad1acatalyticallyinactivedeubiquitinasethatisbroadlyrequiredforpremrnasplicing