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

   

Authors:	Jacewicz, A.; Schwer, B.; Smith, P.; Shuman, S.
Article 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 trypsinresistant 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 center dot 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 center dot Mg2+ is not poised for catalysis in the open domain conformation. Guided by the Prp28 center dot AMPPNP structure, and that of the Drosophila Vasa center dot AMPPNP center dot Mg2+center dot 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. Overexpression of defective ATP-site mutants, but not defective RNA-site mutants, elicited severe dominant-negative growth defects.
Keywords:	helicase; spliceosome; reveals; binding; complex; subunit; genetic interactions; protein prp28p; u1 snrna; q-motif
Journal Title:	Nucleic Acids Research
Volume:	42
Issue:	20
ISSN:	0305-1048
Publisher:	Oxford University Press
Date Published:	2014-11-10
Start Page:	12885
End Page:	12898
Language:	English
ACCESSION:	WOS:000347693200049
DOI:	10.1093/nar/gku930
PROVIDER:	wos
PMCID:	PMC4227776
PUBMED:	25303995
Notes:	Article -- Source: Wos

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