Mutational Analysis of the Yeast DEAH-Box Splicing Factor Prp16

Corresponding author: Beate Schwer, Department of Microbiology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021, bschwer{at}mail.med.cornell.edu (E-mail).

Communicating editor: A. P. MITCHELL

Prp16 is an essential yeast splicing factor that catalyzes RNA-dependent hydrolysis of nucleoside triphosphates. Prp16 is a member of the DEAH-box protein family, which is defined by six collinear sequence motifs. The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis. Mutant alleles of PRP16 were tested for in vivo function by complementation of a prp16 null strain. In motif I (GETGSGKT), alanine substitutions at Gly-378, Lys-379, and Thr-380 were lethal, whereas replacement of the amino acids in positions 373–377 were viable. In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viable. The S505A and T507A mutants in motif III (SAT) were viable. In motif VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A were lethal, whereas G691A, P695A, and G696A supported growth. Instructive structure-function relationships were established by conservative substitutions at essential residues identified by alanine scan. Overexpression of nonviable alleles impaired the growth of wild-type PRP16 cells. Deletion analysis of the 1071-amino-acid Prp16 protein revealed that the N-terminal 204 amino acids and the C-terminal 100 residues were dispensable for PRP16 function in vivo. These studies provide an instructive framework for functional analysis of other DEAH-box splicing factors.

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