Functional characterization of the eukaryotic-specific conserved features of nuclear RNase P in the yeast <italic>Saccharomyces cerevisiae</italic>.

Abstract

RNase P is an endoribonuclease responsible for cleavage of the 5' leader of precursor tRNAs (pre-tRNAs). Most forms of RNase P are ribonucleoprotein complexes containing RNA and protein subunits. Bacterial RNase P contains one RNA moiety and one protein subunit, with the RNA subunit by itself capable of cleaving pre-tRNAs in vitro. Eukaryotic nuclear RNase P also has a single RNA molecule, but many more protein subunits. The RNA subunits are relatively conserved in all three kingdoms of life, but structural elements of the RNA are identified that are conserved only in eukaryotes. Eukaryotic RNase P is closely related to another ribonucleoprotein enzyme, RNase MRP, which is involved in 5.8S rRNA maturation. Several protein subunits are common to RNases P and MRP. My thesis research focuses on functional characterization of the eukaryotic-specific features of nuclear RNase P in the yeast <italic>Saccharomyces cerevisiae </italic>. This work is illustrated in Chapters II and III of my dissertation. Chapter II describes characterization of the eukaryotic-specific conserved elements of the yeast RNase P RNA. Four eukaryotic-specific regions were mutated, and effects of the mutations on cell growth, enzyme function and biogenesis of RNase P were examined. Results suggest that most eukaryotic-specific conserved regions of RNase P RNA are important for holoenzyme assembly, localization and pre-tRNA processing. Chapter III focuses on characterization of the largest common protein subunit of yeast RNase P and MRP, Pop1p. Sequence alignment of Pop1p with its homologs has revealed four conserved regions. Mutations at the highly conserved positions were obtained by randomization mutagenesis, and were screened for conditional growth defects. Most Pop1p mutations affect both pre-tRNA and 5.8S rRNA processing, but a few mutations preferentially weaken only one of the two activities. In many cases, functional defects of RNase P and MRP are consistent with assembly defects of the holoenzymes. Mutations were also obtained that slow RNase P and MRP functions without notably affecting holoenzyme assembly. Taken together this study suggests that Pop1p plays a role in assembly and stability of the RNases P and MRP holoenzymes, but that it also has additional roles in the functions of the enzymes.