An active precursor in assembly of yeast nuclear ribonuclease P.

The RNA-protein subunit assembly of nuclear RNase P was investigated by specific isolation and characterization of the precursor and mature forms of RNase P using an RNA affinity ligand. Pre-RNase P was as active in pre-tRNA cleavage as mature RNase P, although it contained only seven of the nine proteins found in mature RNase P. Pop3p and Rpr2p were not required for maturation of the RPR1 RNA subunit and virtually absent from pre-RNase P, implying that they are dispensable for pre-tRNA substrate recognition and cleavage. The RNase P subunit assembly is likely to occur in the nucleolus, where both precursor and mature forms of RNase P RNA are primarily localized. The results provide insight into assembly of nuclear RNase P, and suggest pre-tRNA substrate recognition is largely determined by the RNA subunit.

Interactions among the protein and RNA subunits of Saccharomyces cerevisiae nuclear RNase P.

Ribonuclease P (RNase P) is a ubiquitous endoribonuclease that cleaves precursor tRNAs to generate mature 5' termini. Although RNase P from all kingdoms of life have been found to have essential RNA subunits, the number and size of the protein subunits ranges from one small protein in bacteria to at least nine proteins of up to 100 kDa. In Saccharomyces cerevisiae nuclear RNase P, the enzyme is composed of ten subunits: a single RNA and nine essential proteins. The spatial organization of these components within the enzyme is not yet understood. In this study we examine the likely binary protein-protein and protein-RNA subunit interactions by using directed two- and three-hybrid tests in yeast. Only two protein subunits, Pop1p and Pop4p, specifically bind the RNA subunit. Pop4p also interacted with seven of the other eight protein subunits. The remaining protein subunits all showed one or more specific protein-protein interactions with the other integral protein subunits. Of particular interest was the behavior of Rpr2p, the only protein subunit found in RNase P but not in the closely related enzyme, RNase MRP. Rpr2p interacts strongly with itself as well as with Pop4p. Similar interactions with self and Pop4p were also detected for Snm1p, the only unique protein subunit so far identified in RNase MRP. This observation is consistent with Snm1p and Rpr2p serving analogous functions in the two enzymes. This study provides a low-resolution map of the multisubunit architecture of the ribonucleoprotein enzyme, nuclear RNase P from S. cerevisiae.