Unraveling The Role of Helicases in Nonfunctional rRNA Decay

rRNAs are essential, long‐lived, and highly abundant, comprising up to 90% of total cellular RNA. However, when structurally intact rRNAs in Saccharomyces cerevisiae are functionally compromised, they are instead rapidly degraded through a quality control pathway termed nonfunctional ribosomal RNA decay (NRD). Currently, two mechanistically distinct types of NRD are known: 18S NRD and 25S NRD. 18S NRD is translation dependent and removes rRNAs with loss of function mutations in the small subunit decoding site. The 18S NRD mechanism involves the translation factor‐like protein Dom34 and its binding partner Hbs1. On the other hand, 25S NRD, which occurs prior to subunit joining, is translation independent and removes nonfunctional rRNAs containing mutations in the large subunit peptidyl transferase center. The 25S NRD mechanism involves polyubiquination of proteins associated with a nonfunctional 25S rRNA through the E3 ligase components Rtt101 and Mms1. While some of the proteins that are involved in either 18S or 25S NRD are known, we have not yet elucidated the complete molecular mechanism of NRD. Since RNA helicases have been implicated in other RNA degradation pathways, we hypothesize that one or more helicases may be important for either or both NRD mechanisms. S. cerevisiae contains 44 different helicases, 14 of which are non‐essential. Thus, we are initially investigating whether any of these 14 helicases are involved in NRD using single gene deletion yeast strains. We transformed BY4741 and the 14 non‐essential helicase knock‐out strains with various rRNA expression plasmids that encode for functional and/or nonfunctional versions of the 18S and 25S rRNAs. These plasmid‐derived rRNAs also contain short, unique sequence tags that allows for specific detection of these rRNAs in the presence of total cellular rRNA. We measured the growth phenotypes of these transformed strains at various temperatures in liquid cultures. At standard temperatures, we see a slow growth phenotype in some of the deletion strains transformed with either nonfunctional 18S or 25S rRNA expression plasmids. Since some helicase deletion strains exhibit either cold‐ or temperature‐sensitivities, we are currently measuring growth phenotypes at these normally restrictive temperatures. In addition, we are measuring the rRNA expression levels in these deletion strains using an RT‐qPCR assay to quantify the level of NRD activity. We hope this work will provide insight into the role of helicases in RNA degradation mechanisms and contribute to our understanding of NRD. Support or Funding Information This work is supported by the Howard Hughes Medical Institute.