Genome-wide search for yeast RNase P substrates reveals role in maturation of intron-encoded box C/D small nucleolar RNAs.
Ribonuclease P (RNase P) is an essential endonuclease responsible for the 5'-end maturation of precursor tRNAs. Bacterial RNase P also processes precursor 4.5S RNA, tmRNA, 30S preribosomal RNA, and several reported protein-coding RNAs. Eukaryotic nuclear RNase P is far more complex than in the bacterial form, employing multiple essential protein subunits in addition to the catalytic RNA subunit. RNomic studies have shown that RNase P binds other RNAs in addition to tRNAs, but no non-tRNA substrates have previously been identified. Additional substrates were identified by using a multipronged approach in the budding yeast Saccharomyces cerevisiae. First, RNase P-dependant changes in RNA abundance were examined on whole-genome microarrays by using strains containing temperature sensitive (TS) mutations in two of the essential RNase P subunits, Pop1p and Rpr1r. Second, RNase P was rapidly affinity-purified, and copurified RNAs were identified by using a genome-wide microarray. Third, to identify RNAs that do not change abundance when RNase P is depleted but accumulate as larger precursors, >80 potential small RNA substrates were probed directly by Northern blot analysis with RNA from the RNase P TS mutants. Numerous potential substrates were identified, of which we characterized the box C/D intron-encoded small nucleolar RNAs (snoRNAs), because these both copurify with RNase P and accumulate larger forms in the RNase P temperature-sensitive mutants. It was previously known that two pathways existed for excising these snoRNAs, one using the pre-mRNA splicing path and the other that was independent of splicing. RNase P appears to participate in the splicing-independent path for the box C/D intron-encoded snoRNAs.
Coughlin
Daniel J
DJ
Department of Biological Chemistry, University of Michigan, 1150 West Medical Center Drive, 3200 MSRB III, Ann Arbor, MI 48109-0606, USA.
Pleiss
Jeffrey A
JA
Walker
Scott C
SC
Whitworth
Gregg B
GB
Engelke
David R
DR
D016681:Genome, Fungal; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D012326:RNA Splicing; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D043262:Ribonuclease P / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D020537:RNA, Small Nucleolar; D043262:Ribonuclease P