Cryo-EM structure of catalytic ribonucleoprotein RNase MRP

Yeast mitochondrial DNA contains multiple promoters that sponsor different levels of transcription. Several promoters are individually located immediately adjacent to presumed origins of replication and have been suggested to play a role in priming of DNA replication. Although yeast mitochondrial DNA replication origins have not been extensively characterized at the primary sequence level, a common feature of these putative origins is the occurrence of a short guanosine-rich region in the priming strand downstream of the transcriptional start site. This situation is reminiscent of vertebrate mitochondrial DNA origins and raises the possibility of common features of origin function. In the case of human and mouse cells, there exists an RNA processing activity with the capacity to cleave at a guanosine-rich mitochondrial RNA sequence at an origin; we therefore sought the existence of a yeast endoribonuclease that had such a specificity. Whole cell and mitochondrial extracts of Saccharomyces cerevisiae contain an RNase that cleaves yeast mitochondrial RNA in a site-specific manner similar to that of the human and mouse RNA processing activity RNase MRP. The exact location of cleavage within yeast mitochondrial RNA corresponds to a mapped site of transition from RNA to DNA synthesis. The yeast activity also cleaved mammalian mitochondrial RNA in a fashion similar to that of the mammalian RNase MRPs. The yeast endonuclease is a ribonucleoprotein, as judged by its sensitivity to nucleases and proteinase, and it was present in yeast strains lacking mitochondrial DNA, which demonstrated that all components required for in vitro cleavage are encoded by nuclear genes. We conclude that this RNase is the yeast RNase MRP.

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

Molecular Biology of the Cell ◽

10.1091/mbc.9.9.2407 ◽

1998 ◽

Vol 9 (9) ◽

pp. 2407-2422 ◽

Cited By ~ 58

Author(s):

Miroslav Dundr ◽

Mark O.J. Olson

Keyword(s):

Molecular Weight ◽

Internal Transcribed Spacer ◽

Rnase P ◽

Rrna Processing ◽

Rnase Mrp ◽

External Transcribed Spacer ◽

Early Anaphase ◽

Late Telophase ◽

Processing Site

Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5′-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5′-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3′-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli.

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A Novel Model for the RNase MRP-Induced Switch Between Different Forms of 5.8S rRNA

10.20944/preprints202104.0765.v1 ◽

2021 ◽

Author(s):

Xiao Li ◽

Janice M Zengel ◽

Lasse Lindahl

Keyword(s):

Rna Polymerase ◽

18S Rrna ◽

Rna Polymerase I ◽

Published Data ◽

Primary Transcript ◽

Rna Molecules ◽

Rnase Mrp ◽

5.8S Rrna ◽

Polymerase I ◽

Novel Model

Processing of the RNA polymerase I pre-rRNA transcript into the mature 18S, 5.8S, and 25S rRNAs requires removing the “spacer” sequences. The canonical pathway for the removal of the ITS1 spacer, located between 18S and 5.8S rRNAs in the primary transcript, involves cleavages at the 3’ end of 18S rRNA and at two sites inside ITS1. The process generates a long and a short 5.8S rRNA that differ in the number of ITS1 nucleotides retained at the 5.8S 5’ end. Here we document a novel pathway that generates the long 5.8S for ITS1 while bypassing cleavage within ITS1. It entails a single endonuclease cut at the 3’-end of 18S rRNA followed by exonuclease Xrn1 degradation of ITS1. Mutations in RNase MRP increase the accumulation of long relative to short 5.8S rRNA; traditionally this is attributed to a decreased rate of RNase MRP cleavage at its target in ITS1, called A3. In contrast, we report here that the MRP induced switch between long and short 5.8S rRNA formation occurs even when the A3 site is deleted. Based on this and our published data, we propose that the switch may depend on RNase MRP processing RNA molecules other than pre-rRNA.

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Efficient site-specific cleavage by RNase MRP requires interaction with two evolutionarily conserved mitochondrial RNA sequences.

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2191 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2191-2201 ◽

Cited By ~ 31

Author(s):

J L Bennett ◽

D A Clayton

Keyword(s):

Sequence Homology ◽

Mammalian Species ◽

Saturation Mutagenesis ◽

Mitochondrial Rna ◽

Rna Sequences ◽

Site Specific ◽

Conserved Sequence ◽

Sequence Block ◽

Rnase Mrp

RNase MRP is a site-specific endonuclease that processes primer mitochondrial RNA from the leading-strand origin of mitochondrial DNA replication. Using deletional analysis and saturation mutagenesis, we have determined the substrate requirements for cleavage by mouse mitochondrial RNase MRP. Two regions of sequence homology among vertebrate mitochondrial RNA primers, conserved sequence blocks II and III, were found to be critical for both efficient and accurate cleavage; a third region of sequence homology, conserved sequence block I, was dispensable. Analysis of insertion and deletion mutations within conserved sequence block II demonstrated that the specificity of RNase MRP accommodates the natural sequence heterogeneity of conserved sequence block II in vivo. Heterologous assays with human RNase MRP and mutated mouse mitochondrial RNA substrates indicated that sequences essential for substrate recognition are conserved between mammalian species.

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