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

Journal Article · · Nucleic Acids Research
DOI:https://doi.org/10.1093/nar/gku930· OSTI ID:1164179
 [1];  [2];  [1];  [1]
  1. Sloan-Kettering Inst., New York, NY (United States)
  2. Weill Cornell Medical College, New York, NY (United States)
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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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Organization:
USDOE
Grant/Contract Number:
AC02-06CH11357
OSTI ID:
1164179
Journal Information:
Nucleic Acids Research, Vol. 42, Issue 20; ISSN 0305-1048
Publisher:
Oxford University PressCopyright Statement
Country of Publication:
United States
Language:
English
Citation Metrics:
Cited by: 20 works
Citation information provided by
Web of Science

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Cited By (7)

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

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