The study of nuclear ribonuclease P in <italic>Saccharomyces cerevisiae</italic> using novel RNA affinity tags.

Abstract

Ribonuclease P (RNase P) is the enzyme responsible for a cleavage of the 5<super>'</super> leaders of pre-tRNAs in all living organisms. In the yeast <italic>Saccharomyces cerevisiae</italic>, nuclear RNase P consists of one RNA (<italic>RPR1</italic> RNA) and nine protein subunits. <italic> RPR1</italic> RNA is initially transcribed as a precursor and subsequently processed into a mature form. Defects in subunit assembly have been associated with a failure of <italic>RPR1</italic> RNA to be processed from the precursor to mature form. In order to understand the holoenzyme subunit assembly, the studies in this dissertation address the nature of the precursor form of RNase P (pre-RNase P). Instead of using a long, multistep chromatographic purification of RNase P, which could lead to degradation and loss of the enzymatic activity, a rapid and specific affinity isolation of the enzyme is strongly preferred. However, the RNA affinity ligands for detection and isolation of RNAs or ribonucleoproteins have been lacking. In this study, two small RNA tags have been identified through in vitro selection. These tags bind specifically to Sephadex or streptavidin, and the binding to their targets can be disrupted under mild conditions with either dextran or d-biotin, respectively. This useful feature allows specific isolation and recovery of the tagged RNAs or ribonucleoproteins with intact structures and activity. To isolate RNase P, <italic>RPR1</italic> RNA was tagged at various positions with the Sephadex-binding RNA, enabling a rapid and specific affinity isolation of either pre- or mature-RNase P. Pre-RNase P was found to be as active in pre-tRNA cleavage in vitro as mature-RNase P. Its protein subunit composition was also similar to that of mature-RNase P, except that very little Pop3p and Rpr2p could be detected relative to the other subunits. Further depletion of these two subunits did not affect <italic>RPR1</italic> RNA maturation even though they are required for RNase P function in vivo. In situ hybridization showed that pre-RNase P RNA was localized primarily in the nucleolus, indicating that the assembly is likely to occur there. The finding that pre-RNase P was enzymatically active suggests that, in addition to being the precursor, it might have an in vivo catalytic role for either pre-tRNAs or other unidentified substrates.