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Title: Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28

Abstract

Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.

Authors:
 [1];  [2];  [1];  [1]
  1. Sloan-Kettering Inst., New York, NY (United States)
  2. Weill Cornell Medical College, New York, NY (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1164179
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Volume: 42; Journal Issue: 20; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Jacewicz, Agata, Schwer, Beate, Smith, Paul, and Shuman, Stewart. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28. United States: N. p., 2014. Web. doi:10.1093/nar/gku930.
Jacewicz, Agata, Schwer, Beate, Smith, Paul, & Shuman, Stewart. Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28. United States. https://doi.org/10.1093/nar/gku930
Jacewicz, Agata, Schwer, Beate, Smith, Paul, and Shuman, Stewart. 2014. "Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28". United States. https://doi.org/10.1093/nar/gku930. https://www.osti.gov/servlets/purl/1164179.
@article{osti_1164179,
title = {Crystal structure, mutational analysis and RNA-dependent ATPase activity of the yeast DEAD-box pre-mRNA splicing factor Prp28},
author = {Jacewicz, Agata and Schwer, Beate and Smith, Paul and Shuman, Stewart},
abstractNote = {Yeast Prp28 is a DEAD-box pre-mRNA splicing factor implicated in displacing U1 snRNP from the 5' splice site. Here we report that the 588-aa Prp28 protein consists of a trypsin-sensitive 126-aa N-terminal segment (of which aa 1–89 are dispensable for Prp28 function in vivo) fused to a trypsin-resistant C-terminal catalytic domain. Purified recombinant Prp28 and Prp28-(127–588) have an intrinsic RNA-dependent ATPase activity, albeit with a low turnover number. The crystal structure of Prp28-(127–588) comprises two RecA-like domains splayed widely apart. AMPPNP•Mg2+ is engaged by the proximal domain, with proper and specific contacts from Phe194 and Gln201 (Q motif) to the adenine nucleobase. The triphosphate moiety of AMPPNP•Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28•AMPPNP structure, and that of the Drosophila Vasa•AMPPNP•Mg2+•RNA complex, we targeted 20 positions in Prp28 for alanine scanning. ATP-site components Asp341 and Glu342 (motif II) and Arg527 and Arg530 (motif VI) and RNA-site constituent Arg476 (motif Va) are essential for Prp28 activity in vivo. Synthetic lethality of double-alanine mutations highlighted functionally redundant contacts in the ATP-binding (Phe194-Gln201, Gln201-Asp502) and RNA-binding (Arg264-Arg320) sites. As a result, overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.},
doi = {10.1093/nar/gku930},
url = {https://www.osti.gov/biblio/1164179}, journal = {Nucleic Acids Research},
issn = {0305-1048},
number = 20,
volume = 42,
place = {United States},
year = {Fri Oct 10 00:00:00 EDT 2014},
month = {Fri Oct 10 00:00:00 EDT 2014}
}

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Works referenced in this record:

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Crystal Structure of Prp5p Reveals Interdomain Interactions that Impact Spliceosome Assembly
journal, December 2013


Reconstitution of both steps of Saccharomyces cerevisiae splicing with purified spliceosomal components
journal, November 2009


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journal, December 2000


Prp43 Is an Essential RNA-dependent ATPase Required for Release of Lariat-Intron from the Spliceosome
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Works referencing / citing this record:

Domain Requirements and Genetic Interactions of the Mud1 Subunit of the Saccharomyces cerevisiae U1 snRNP
journal, November 2018


Domain Requirements and Genetic Interactions of the Mud1 Subunit of the Saccharomyces cerevisiae U1 snRNP
journal, November 2018


Mechanism of 5ʹ splice site transfer for human spliceosome activation
journal, April 2019


Understanding pre-mRNA splicing through crystallography
journal, August 2017


Molecular architecture of the human U4/U6.U5 tri-snRNP
journal, February 2016


Structures of the fully assembled Saccharomyces cerevisiae spliceosome before activation
journal, May 2018


Prespliceosome structure provides insights into spliceosome assembly and regulation
journal, July 2018


Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1
journal, February 2015