AN RNASE-P RNA SUBUNIT MUTATION AFFECTS RIBOSOMAL-RNA PROCESSING

RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms, While analyzing mutat ions in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of similar to 193 nucleoti des was observed, This abundant RNA was identified as a 3' extended fo rm of the 5.8S rRNA, This strain also displays a slightly elevated lev el of other rRNA processing intermediates with 5'-ends at processing s ite A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8 S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partia lly purified to remove contaminating nucleases, Cleavage assays were p erformed using an rRNA substrate transcribed in vitro which includes t he 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the pea k fractions of nuclear RNase P, but not with fractions corresponding t o mitochondrial RNase P or ribonuclease MRP RNA, The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase P. The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRN A processing pathway are considered. These observations suggest an int imate relationship between the processes of tRNA and rRNA maturation i n the eukaryotic nucleus.