Ribosomal Protein S14 of Saccharomyces cerevisiae Regulates Its Expression by Binding to RPS14B Pre-mRNA and to 18S rRNA

Abstract

Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B. Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14Bregulation. Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity. These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA. Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition. D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target. Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits. The emetine-resistant protein, rpS14-Em^RR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays. This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure.

ACKNOWLEDGMENTS

We thank Rachel Green, Javier Lopez, Jon Warner, Josep Vilardell, and colleagues in our laboratory for critical reading of the manuscript. We are grateful to Dhruba SenGupta, Beilin Zhang, and Marvin Wickens for sharing the three-hybrid system and for advice on using the system. We also thank Chuck Allerson and Susan Liebman for providing the templates for transcription of the IRE and yeast 18S rRNA, respectively, and Robin Gutell for 16S and 18S rRNA structure predictions.

This work was supported by U.S. Public Health Service research grant GM28301 to J.L.W.