Comprehensive Mutational Analysis of Yeast DEXD/H Box RNA Helicases Required for Small Ribosomal Subunit Synthesis

Abstract

The 17 putative RNA helicases required for pre-rRNA processing are predicted to play a crucial role in ribosome biogenesis by driving structural rearrangements within preribosomes. To better understand the function of these proteins, we have generated a battery of mutations in five putative RNA helicases involved in 18S rRNA synthesis and analyzed their effects on cell growth and pre-rRNA processing. Our results define functionally important residues within conserved motifs and demonstrate that lethal mutations in predicted ATP binding-hydrolysis motifs often confer a dominant negative phenotype in vivo when overexpressed in a wild-type background. We show that dominant negative mutants delay processing of the 35S pre-rRNA and cause accumulation of pre-rRNA species that normally have low steady-state levels. Our combined results establish that not all conserved domains function identically in each protein, suggesting that the RNA helicases may have distinct biochemical properties and diverse roles in ribosome biogenesis.

We are grateful to Alicia Lee, Paul Stone, and Neal Janson for expert technical assistance. We thank the members of our laboratory for critically reading the manuscript.

This work was supported by Leslie H. Warner and Anna Fuller Postdoctoral Cancer Research fellowships (S.G.) and the National Institutes of Health (S.J.B.; GM52581).