Mutational analysis of the RNA helicase Dhh1 in Ste12 expression and yeast mating.

Abstract

Dhh1 and Dhh1 homologues (RCK/p54/DDX6) are members of the DEAD-box protein family of RNA helicases. These proteins display conserved sequence motifs for ATPase and RNA binding activities. Dhh1 is a component of the P-bodies (processing bodies) of mRNA granules and functions as an mRNA decapping activator in Saccharomyces cerevisiae. Dhh1 also contributes to gene-specific regulation during yeast mating. The dhh1 deletion mutation results in a significant decrease in the expression of Ste12, a mating-specific transcription factor, showing severe mating defects. Here, we introduced amino-acid substitution mutations in the ATPase and RNA binding domains of Dhh1 and also constructed a deletion of 79 amino acids at the Q/P-rich C-terminal region. The mutations in ATPase A and B motif (K96R, D195A) and C-terminus deletion showed reduced levels of mating efficiency as well as Ste12 protein expression. The Q/P-rich C-terminal region of Dhh1 was dispensable for growth at nonpermissive temperature 37°C but appeared to play an important role in regulating the Ste12 protein expression and mating processes. The P-body accumulation induced by treatment with α-mating factor required ATPase, RNA-binding and the Q/P-rich C-terminal domains of Dhh1.