MUTATIONAL ANALYSIS OF THE YEAST DEAH-BOX SPLICING FACTOR PRP16

Prp16 is an essential yeast splicing factor that catalyzes RNA-depende nt hydrolysis of nucleoside triphosphates. Prp16 is a member of the DE AH-box protein family, which is defined by six collinear sequence moti fs. The importance of residues within four of the conserved motifs was assessed by alanine-scanning mutagenesis. Mutant alleles of PRP16 wer e tested for in vivo function by complementation of a Delta 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 i n positions 373-377 were viable. In the signature DEAH-box (motif II), Asp-473 and Glu-474 were essential, whereas the H476A mutant was viab le. The S505A and T507A mutants in motif III (SAT) were viable. In mot if VI (QRSGRAGRTAPG), mutants Q685A, R686A, G688A, R689A, and R692A we re lethal, whereas G691A, P695A, and G696A supported growth. Instructi ve structure-function relationships were established by conservative s ubstitutions at essential residues identified by alanine scan. Overexp ression of nonviable alleles impaired the growth of wild-type PRP16 ce lls. Deletion analysis of the 1071-amino-acid Prp16 protein revealed t hat the N-terminal 204 amino acids and the C-terminal 100 residues wer e dispensable for PRP16 function in vivo. These studies provide an ins tructive framework for functional analysis of other DEAH-box splicing factors.