RNase MRP is a eukaryotic endoribonuclease involved in ribosomal RNA processing. It cleaves the rRNA primary transcript in a site-specific manner to generate mature 5.8S rRNA. In Saccharomyces cerevisiae, RNase MRP is composed of one RNA subunit and at least nine protein subunits. One of the largest protein subunits is Pop4p, a 32.9 kD protein. The RNA molecule is encoded by the single copy gene, RPP2 (Ribosomal RNA Processing 2).

The details of the interactions between the RNA and protein components of RNase MRP are not known, although immunoprecipitation studies indicate that Pop4p is associated with RNase MRP RNA. My project is to map the binding site of Pop4p to RNase MRP RNA by purifying of Pop4p and using gel shift assay.

In order to purify Pop4, it must be overexpressed. However, previous researchers have found that Pop4p is highly insoluble and precipitates out of solution. An archeal homologue of Pop4p, Mth Rpp29, found in the closely related enzyme, RNase P, is soluble and its structure has been determined. Mth Rpp29 consists only of a conserved core sequence of amino acids that is found in Pop4p isolated from all species. Therefore, we constructed a mutant Pop4 gene, consisting only of the “conserved core”. Currently, I am working on inserting the wild type Pop4 and mutant Pop4 gene into a suitable vector to allow overexpression, to be followed by purification using Immobilized metal Affinity Chromatography. I am also working on creating other mutant versions of Pop4p, where certain predicted secondary structures from the non-conserved region are deleted, which although not functional may be purified and used for binding assays.