RNase MRP is required for entry of 35S precursor rRNA into the canonical processing pathway.
RNase MRP is a nucleolar RNA-protein enzyme that participates in the processing of rRNA during ribosome biogenesis. Previous experiments suggested that RNase MRP makes a nonessential cleavage in the first internal transcribed spacer. Here we report experiments with new temperature-sensitive RNase MRP mutants in Saccharomyces cerevisiae that show that the abundance of all early intermediates in the processing pathway is severely reduced upon inactivation of RNase MRP. Transcription of rRNA continues unabated as determined by RNA polymerase run-on transcription, but the precursor rRNA transcript does not accumulate, and appears to be unstable. Taken together, these observations suggest that inactivation of RNase MRP blocks cleavage at sites A0, A1, A2, and A3, which in turn, prevents precursor rRNA from entering the canonical processing pathway (35S > 20S + 27S > 18S + 25S + 5.8S rRNA). Nevertheless, at least some cleavage at the processing site in the second internal transcribed spacer takes place to form an unusual 24S intermediate, suggesting that cleavage at C2 is not blocked. Furthermore, the long form of 5.8S rRNA is made in the absence of RNase MRP activity, but only in the presence of Xrn1p (exonuclease 1), an enzyme not required for the canonical pathway. We conclude that RNase MRP is a key enzyme for initiating the canonical processing of precursor rRNA transcripts, but alternative pathway(s) might provide a backup for production of small amounts of rRNA.
Lindahl
Lasse
L
Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, Maryland 21250, USA. lindahl@umbc.edu
Bommankanti
Ananth
A
Li
Xing
X
Hayden
Lauren
L
Jones
Adrienne
A
Khan
Miriam
M
Oni
Tolulope
T
Zengel
Janice M
JM
D001483:Base Sequence; D015152:Blotting, Northern; D004722:Endoribonucleases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010641:Phenotype; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000235:genetics / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014158:Transcription, Genetic
D012322:RNA Precursors; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D004722:Endoribonucleases; D005095:Exoribonucleases; C058330:mitochondrial RNA-processing endoribonuclease; C068398:XRN1 protein, S cerevisiae