Binding and cleavage of unstructured RNA by nuclear RNase P

Marvin, Michael C.; Walker, Scott C.; Fierke, Carol A.; Engelke, David R.

doi:10.1261/rna.2633611

Cited by 17 publications

(28 citation statements)

References 50 publications

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“…Based on our data (Fig. 3) several RNase P proteins are positioned to interact with substrates: Pop1, Pop4, Pop5, and, potentially, Rpp1, consistent with recent results of enzyme-substrate crosslinking studies (Marvin et al 2011b). …”

Section: D Mapping Of Rna-protein Interactions In Yeast Rnase Psupporting

confidence: 91%

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

Khanova¹,

Esakova²,

Perederina³

et al. 2012

RNA

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Eukaryotic ribonuclease (RNase) P and RNase MRP are closely related ribonucleoprotein complexes involved in the metabolism of various RNA molecules including tRNA, rRNA, and some mRNAs. While evolutionarily related to bacterial RNase P, eukaryotic enzymes of the RNase P/MRP family are much more complex. Saccharomyces cerevisiae RNase P consists of a catalytic RNA component and nine essential proteins; yeast RNase MRP has an RNA component resembling that in RNase P and 10 essential proteins, most of which are shared with RNase P. The structural organizations of eukaryotic RNases P/MRP are not clear. Here we present the results of RNA-protein UV crosslinking studies performed on RNase P and RNase MRP holoenzymes isolated from yeast. The results indicate locations of specific protein-binding sites in the RNA components of RNase P and RNase MRP and shed light on the structural organizations of these large ribonucleoprotein complexes.

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Section: D Mapping Of Rna-protein Interactions In Yeast Rnase Psupporting

confidence: 91%

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

Khanova¹,

Esakova²,

Perederina³

et al. 2012

RNA

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“…Confirming that these RNAs are RNase P substrates in vivo is difficult given that in vitro studies (Chamberlain et al 1996;Coughlin et al 2008;Marvin et al 2011) have shown that nuclear RNase P can bind and cleave singlestranded RNA at multiple positions without clear sequence or structural requirements. Our in vitro study of cleavage preferences deliberately chose both sense and antisense RNAs across a region where the balance was affected by RNase P mutations, showing that RNase P could bind all segments across the region and cleave RNAs from both strands at multiple places (Camblong et al 2007;Coughlin et al 2008;Marvin et al 2011).…”

Section: Discussionmentioning

confidence: 99%

“…Like its bacterial counterpart, in addition to pre-tRNA, other possible substrates have been identified in eukaryotes (Chamberlain et al 1996;Yang and Altman 2007;Coughlin et al 2008;Wilusz et al 2008). In addition, purified nuclear RNase P's affinity for single-stranded RNA and its ability to cleave at multiple sites in vitro without obvious sequence specificity (see Marvin et al 2011) indicate a broad capacity to serve as a relatively nonspecific RNA endonuclease.…”

Section: Introductionmentioning

confidence: 99%

Accumulation of noncoding RNA due to an RNase P defect in Saccharomyces cerevisiae

Marvin

Clauder‐Münster²,

Walker

et al. 2011

RNA

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Ribonuclease P (RNase P) is an essential endoribonuclease that catalyzes the cleavage of the 59 leader of pre-tRNAs. In addition, a growing number of non-tRNA substrates have been identified in various organisms. RNase P varies in composition, as bacterial RNase P contains a catalytic RNA core and one protein subunit, while eukaryotic nuclear RNase P retains the catalytic RNA but has at least nine protein subunits. The additional eukaryotic protein subunits most likely provide additional functionality to RNase P, with one possibility being additional RNA recognition capabilities. To investigate the possible range of additional RNase P substrates in vivo, a strand-specific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting that nuclear RNase P participates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcripts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either compromised splicing or failure to efficiently turn over pre-mRNAs that do not enter the splicing pathway. Taken together with the high complexity of the nuclear RNase P holoenzyme and its relatively nonspecific capacity to bind and cleave mixed sequence RNAs, these data suggest that nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in pre-tRNA biogenesis.

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“…Poly(U) 50 RNA was purchased from Integrated DNA Technology. The smaller sizes of poly(U) RNA were generated by alkaline hydrolysis and subsequent purification by passive elution from denaturing polyacrylamide gels (Marvin et al 2011). …”

Section: Rna Preparationmentioning

confidence: 99%

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

Pai

Kaplan

Kweon

et al. 2014

RNA

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Many RNAs are known to act as regulators of transcription in eukaryotes, including certain small RNAs that directly inhibit RNA polymerases both in prokaryotes and eukaryotes. We have examined the potential for a variety of RNAs to directly inhibit transcription by yeast RNA polymerase II (Pol II) and find that unstructured RNAs are potent inhibitors of purified yeast Pol II. Inhibition by RNA is achieved by blocking binding of the DNA template and requires binding of the RNA to Pol II prior to open complex formation. RNA is not able to displace a DNA template that is already stably bound to Pol II, nor can RNA inhibit elongating Pol II. Unstructured RNAs are more potent inhibitors than highly structured RNAs and can also block specific transcription initiation in the presence of basal transcription factors. Crosslinking studies with ultraviolet light show that unstructured RNA is most closely associated with the two large subunits of Pol II that comprise the template binding cleft, but the RNA has contacts in a basic residue channel behind the back wall of the active site. These results are distinct from previous observations of specific inhibition by small, structured RNAs in that they demonstrate a sensitivity of the holoenzyme to inhibition by unstructured RNA products that bind to a surface outside the DNA cleft. These results are discussed in terms of the need to prevent inhibition by RNAs, either though sequestration of nascent RNA or preemptive interaction of Pol II with the DNA template.

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Binding and cleavage of unstructured RNA by nuclear RNase P

Cited by 17 publications

References 50 publications

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

Structural organizations of yeast RNase P and RNase MRP holoenzymes as revealed by UV-crosslinking studies of RNA–protein interactions

Accumulation of noncoding RNA due to an RNase P defect in Saccharomyces cerevisiae

RNAs nonspecifically inhibit RNA polymerase II by preventing binding to the DNA template

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