An RNase P RNA subunit mutation affects ribosomal RNA processing

Abstract

RNase P is a ribonucleoprotein endoribonuclease responsible for the 5' maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of approximately 193 nucleotides was observed. This abundant RNA was identified as a 3'extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to 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 R The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.

Partially Processed pre-rRNA Is Preserved in Association with Processing Components in Nucleolus-derived Foci during Mitosis

“…RNase MRP is implicated in the cleavage of pre-rRNA at site A3 within ITS1 (Lygerou et al, 1994). RNase P participates in cleavages in the 5Ј-ETS, ITS1, and ITS2 of the primary 35S pre-rRNA transcript in yeast (Chamberlain et al, 1996;Stolc and Altman, 1997). Thus, it seems likely that nucleolar endonucleases remain associated with nascent prerRNA, which further supports the idea that multiple processing components are contained in complexes whose overall organization is preserved during mitosis.…”

“…The endoribonuclease MRP cleaves the yeast pre-rRNA at site A 3 within the ITS1 region of pre-rRNA (Schmitt and Clayton, 1992;Chu et al, 1994;Lygerou et al, 1996a), and endoribonuclease RNase P plays a role in processing of the 5Ј-ETS, ITS1, and ITS2 regions of pre-rRNA (Chamberlain et al, 1996;Stolc and Altman, 1997). Both enzymes are structurally and functionally related and share at least three protein subunits, Pop1, Pop3p, and Pop4p (Tollervey and Kiss, 1997 and references therein).…”

Global identification of noncoding RNAs in Saccharomyces cerevisiae by modulating an essential RNA processing pathway

“…All of the known ncRNAs (22)(23)(24)(25)(26)(27), encompassing a large spectrum of tRNA, rRNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), RNase P, RNase MRP, and signal recognition particle components, showed strong or time-varying RNA transcription signals (Fig. 6, which is published as supporting information on the PNAS web site).…”

Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum

“…The aforementioned sequencing approach confirms the 5Ј end of the 5.8 S rRNA also for the AX4 strain, where the 3Ј end, however, apparently shows some heterogeneity with molecules being 1, 2, 4 and once also 6 nucleotides shorter (data not shown). In most organisms, there is a long and a short form of the 5.8 S rRNA that vary in yeast by 7 nts at the 5Ј end (30). To confirm the absence of a second 5.8 S rRNA in D. discoideum, we performed a primer extension analysis.…”

“…B, sequence comparison of the 5Ј end of 5.8 S rRNA from D. discoideum (D.d.) and the long (L) and short (S) form of S. cerevisiae (S.c.), using published sequences (30).…”