A nuclear function for RNase MRP.

The site-specific endoribonuclease RNase MRP was first identified 7 years ago as an RNA-processing activity in mammalian cells (1, 2). In the original description of this ribonucleoprotein, as isolated from either mouse or human cells, it was shown that the enzyme had the capacity to cleave RNA in a sequence-specific manner that matched one of the transition sites from RNA to DNA synthesis at the origin of leading-strand replication in mitochondrial DNA (mtDNA). It was soon learned that the RNA portion of this ribonucleoprotein entity was encoded by a nuclear gene. Further work identified the sequences of the mouse (3) and human (4) single-copy genes for the RNA component of the respective RNase MRP enzymes. In the last few years, both RNase MRP enzymatic activity and the identity of the RNase MRP RNA species (MRP RNA) and its respective nuclear gene have been obtained and characterized for Bos taurus (5), Xenopus laevis (6), and the yeast Saccharomyces cerevisiae (7). In all of these cases, the activities appear capable of processing mitochondrial RNA (mtRNA) sequences from both the homologous leading-strand mtDNA origin as well as across species boundaries. However, in the initial and subsequent analyses of intracellular distribution of MRP RNA and enzymatic activity (2, 4, 8), it has been clear that the majority of both is located in the nucleus. Furthermore, the major autoantigen associated with this ribonucleoprotein particle (Th or To), for which patient sera are available for the detection of antigen inside cells, is preferentially located in the nucleolus (9, 10). For these reasons it has been logical to assume that RNase MRP might have an important function in the nucleus, and its apparent predominant localization to the nucleolus has invited continuing speculation that RNase MRP might somehow be involved in nuclear rRNA processing. The possibility of a critical role for RNase MRP in the nucleus was underscored by the finding that the RNA component of this enzyme in S. cerevisiae was essential for viability (7). With the availability of the yeast gene for MRP RNA, the stage was set for a hunt for a nuclear function for this activity. Success came from two routes. In a recent article in the Proceedings, Lindahl and coworkers (11) reported their results of searching for genes which might complement a previously characterized mutant of S. cerevisiae that displayed an abnormal rRNA processing pattern. Their earlier studies with this mutant (rrp2-2) demonstrated that an altered pattern of rRNA processing was present that exhibited itself as both an increased amount of some precursor rRNA forms and an alteration in the stoichiometry of 5.8S rRNAs, the formation of which occurs at mid to late stages in the overall rRNA processing pathway (12). In searching for the basis of the altered processing pattern (11), it was shown that the genetic defect responsible for the faulty processing phenotype was in the NMEJ gene, previously reported to encode S. cerevisiae MRP RNA (7). Once implicated, the NMEJ gene was subjected to site-directed mutational analysis by deletion of bases within the gene from a midpoint restriction site. Small deletions from this site had no effect on rescue of rRNA processing or growth patterns, whereas larger deletions affected cell viability at the restrictive temperature while also failing to rescue abnormal processing. These data were taken to confirm the initial conclusion that the rrp2-2 mutation was indeed complemented by the NMEJ gene. The mutation was further characterized by sequencing the NMEJ gene in both wildtype and mutant strains. Wild-type sequence was as reported (7), whereas the NMEJ sequence in the rrp2-2 mutant contained a single G-to-A transition at nucleotide position 122. The authors tested for the relative stability of the wild-type MRP RNA versus that with a single base change and found that there was no difference in the relative abundance of full-length transcripts that could be isolated from each of the two strains. It was therefore concluded that the most likely explanation for the problem resulting from a single base change in MRP RNA was at the level of catalytic activity of the enzyme and that the defect could not be explained on the basis of differential MRP RNA stability. All of these results are in excellent agreement with the report of Schmitt and Clayton (13) in which the ramifications of altered levels of expression of wild-type MRP RNA or the expression of altered forms of MRP RNA were examined in order to search for a nuclear phenotypic consequence. As the NMEJ gene is essential for cellular viability (7), the experimental strategy was to examine the effects of regulated expression in a strain in which the chromosomal copy of NMEJ had been inactivated and the viability of the cell was rescued by maintenance of a plasmid-borne copy of NMEJ. When NMEJ was down-regulated by glucose repression of expression from the GAL] promoter, there was a significant depletion of the amount of the NMEJ gene product, wild-type intracellular MRP RNA. Concomitant with the decrease in MRP RNA it was found that there was a reversal in the stoichiometry of the two mature forms of 5.8S rRNA; in the MRP RNA-depleted condition, the 7-nucleotide-longer version of 5.8S rRNA was =1O times more abundant than the shorter species lacking this 7-nucleotide sequence at the 5' end. These results were in contrast to the normal stoichiometry in which the shorter version of 5.8S rRNA is %10-fold more abundant than the slightly longer version. Two temperature-conditional mutant strains were examined in which the NMEJ gene had suffered either an internal deletion or a point mutation consisting of an A-to-G transition at position 122 of the RNA sequence. Indeed, this single base mutation, which was selected as a temperature-conditional mutation for growth and is a tight mutation for this phenotype, is identical to the rrp2 mutations isolated independently by Lindahl and coworkers (11, 12) and by Shuai and Warner (14) (rrp2-2 and rrp2-1, respectively). Thus it is likely that this particular position defines a critical location within the MRP RNA sequence with regard to its functional capacity. With both of their conditional mutants, Schmitt and Clayton (13) found a similar phenotypenamely, the altered st6ichiometry of 5.8S rRNA forms. This was most pronounced with the single-base mutation (termed P6; ref. 13); in this case there was no obvious accumulation of larger precursor rRNAs to the extent observed by others (12, 14). However, the clear alteration in the processing pattern of mature 5.8S rRNAs is a result that is consistent between two studies (11, 13), and in each case (11, 13, 14) an accumulation of a slightly larger