Implication of RNases PH in Turnover of M1 RNA

Abstract

Figure 1. Effects on sequestration of C5 protein on M1 RNA stability in endoribonuclease-deficient cells. Total cellular RNA was isolated from strains GW10 (wild type), GW11 (rng‒), and GW20 (rne) containing plasmids at the indicated times after the rifampicin treatment. Each RNA sample (30 μg) was fractionated on a 5% polyacrylamide gel containing 7 M urea and analyzed by northern blotting. A stem P12-specific probe was used as a probe for M1 RNA. Escherichia coli RNase P was initially characterized as a tRNA-processing enzyme that removes extraneous 5' sequences from precursor tRNAs to generate mature 5' termini. In addition to tRNA processing, RNase P is involved in the processing of other non-tRNA substrates (e.g., 4.5S RNA and tmRNA) and the decay of several mRNAs.. The RNase P holoenzyme is composed of two subunits, a large RNA subunit (M1 RNA, 377 nucleotides) and a small basic protein (C5 protein, 119 amino acids). M1 RNA is the catalytic subunit that can perform the reaction as a ribozyme in the absence of C5 protein in vitro, but both the protein and RNA components are essential in vivo. C5 protein stabilizes the catalytically active conformation of M1 RNA and modulates its substrate specificity. Previously, we have shown that the turnover rate of free M1 RNA increases when C5 protein is sequestered by truncated M1 RNA transcripts, such as P12-deleted M1 RNA, in the cell, suggesting that the protein also functions as a metabolic stabilizer of M1 RNA. The size of C5 protein is only one-tenth of M1 RNA. Therefore, it is an interesting issue how this small protein can protect such a large RNA molecule from degradation. To address this issue, in this study, we tried to identify RNases responsible for the rapid turnover of free M1 RNA. For this purpose, we introduced into cells plasmid pLMd12, which overproduced P12deleted M1 RNA derivative as a C5 protein-interacting RNA, so that this plasmid-borne, overexpressed M1 RNA derivative could sequester C5 protein available for M1 RNA binding and thereby generate a protein-free form of M1 RNA within cells. Generally RNA degradation is initiated by endoribonucleolytic cleavage and RNase E plays an important role in this endoribonucleolytic cleavage. Therefore, we investigated the turnover rate of free M1 RNA in strains GW20 and GW11, which showed a temperature-sensitive RNase E phenotype and a RNase G mutant phenotype, respectively. Total RNAs prepared from GW20 and GW11 cells containing plasmid pLMdP12 were analyzed. The cellular levels of M1 RNA level after the treatment of rifampicin that inhibits transcription were monitored by northern blot analysis and then the metabolic stability of M1 RNA was assessed (Fig. 1). We observed no decrease of the M1 RNA stability in both cells. Surprisingly the M1 RNA stability was not decreased in the wild type control strain GW10 either. This contrasts our previous report that the sequestration of C5 protein via overexpression of C5 protein-interacting RNA, such as P12-deleted M1 RNA, reduces the metabolic stability