Co-surveillance of ribosomal RNA by the exosome complex and nucleolar RNAi in C. elegans

AbstractEukaryotic cells express a wide variety of endogenous small regulatory RNAs that function in the nucleus. We previously found that erroneous rRNAs induce the generation of antisense ribosomal siRNAs (risiRNAs) which silence the expression of rRNAs via the nuclear RNAi defective (Nrde) pathway. To further understand the biological roles and mechanisms of this class of small regulatory RNAs, we conducted forward genetic screening to identify factors involved in risiRNA generation in Caenorhabditis elegans. We found that risiRNAs accumulated in the RNA exosome mutants. risiRNAs directed a NRDE-dependent silencing of pre-rRNAs in the nucleolus. In the presence of risiRNA, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNA inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the site of RNAi. Meanwhile, exosome mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results establish a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.

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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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A Conserved NRDE-2/MTR-4 Complex Mediates Nuclear RNAi in Caenorhabditis elegans

Genetics ◽

10.1534/genetics.120.303631 ◽

2020 ◽

Vol 216 (4) ◽

pp. 1071-1085

Author(s):

Gang Wan ◽

Jenny Yan ◽

Yuhan Fei ◽

Daniel J. Pagano ◽

Scott Kennedy

Keyword(s):

Caenorhabditis Elegans ◽

Gene Silencing ◽

Small Interfering Rnas ◽

Interacting Protein ◽

C Elegans ◽

Link Type ◽

Evolutionarily Conserved ◽

Small Regulatory Rnas ◽

Regulatory Rnas ◽

Nuclear Rnai

Small regulatory RNAs, such as small interfering RNAs (siRNAs) and PIWI-interacting RNAs, regulate splicing, transcription, and genome integrity in many eukaryotes. In Caenorhabditis elegans, siRNAs bind nuclear Argonautes (AGOs), which interact with homologous premessenger RNAs to recruit downstream silencing effectors, such as NRDE-2, to direct cotranscriptional gene silencing [or nuclear RNA interference (RNAi)]. To further our understanding of the mechanism of nuclear RNAi, we conducted immunoprecipitation-mass spectrometry on C. elegansNRDE-2. The major NRDE-2 interacting protein identified was the RNA helicase MTR-4. Co-immunoprecipitation analyses confirmed a physical association between NRDE-2 and MTR-4. MTR-4 colocalizes with NRDE-2 within the nuclei of most/all C. elegans somatic and germline cells. MTR-4 is required for nuclear RNAi, and interestingly, MTR-4 is recruited to premessenger RNAs undergoing nuclear RNAi via a process requiring nuclear siRNAs, the nuclear AGO HRDE-1, and NRDE-2, indicating that MTR-4 is a component of the C. elegans nuclear RNAi machinery. Finally, we confirm previous reports showing that human (Hs)NRDE2 and HsMTR4 also physically interact. Our data show that the NRDE-2/MTR-4 interactions are evolutionarily conserved, and that, in C. elegans, the NRDE-2/MTR-4 complex contributes to siRNA-directed cotranscriptional gene silencing.

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New Model for the Yeast RNA Polymerase I Transcription Cycle

Molecular and Cellular Biology ◽

10.1128/mcb.21.15.4847-4855.2001 ◽

2001 ◽

Vol 21 (15) ◽

pp. 4847-4855 ◽

Cited By ~ 44

Author(s):

Pavel Aprikian ◽

Beth Moorefield ◽

Ronald H. Reeder

Keyword(s):

Rna Polymerase ◽

Transcription Initiation ◽

Polymerase Activity ◽

Rna Polymerase I ◽

Rrna Synthesis ◽

New Model ◽

Pol I ◽

Stable Association ◽

Polymerase I ◽

Novel Model

ABSTRACT Using an immobilized template assay, we observed two steps in assembly of the yeast RNA polymerase I (Pol I) preinitiation complex: stable binding of upstream activating factor (UAF) followed by recruitment of Pol I-Rrn3p and core factor (CF). Pol I is required for stable association of CF with the promoter and can be recruited in the absence of Rrn3p. Upon transcription initiation, Pol I-Rrn3p and CF dissociate from the promoter while UAF remains behind. These findings support a novel model in which the Pol I basal machinery cycles on and off the promoter with each round of transcription. This model accounts for previous observations that rRNA synthesis may be controlled by regulating both promoter accessibility and polymerase activity.

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

International Journal of Molecular Sciences ◽

10.3390/ijms22136690 ◽

2021 ◽

Vol 22 (13) ◽

pp. 6690

Author(s):

Xiao Li ◽

Janice M. Zengel ◽

Lasse Lindahl

Keyword(s):

Rna Polymerase ◽

18S Rrna ◽

Canonical Pathway ◽

Rna Polymerase I ◽

Published Data ◽

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 involves cleavages at the 3′ end of 18S rRNA and at two sites inside ITS1. The process can generate either a long or a short 5.8S rRNA that differs in the number of ITS1 nucleotides retained at the 5.8S 5′ end. Here we document a novel pathway to the long 5.8S, which bypasses cleavage within ITS1. Instead, the entire ITS1 is degraded from its 5′ end by exonuclease Xrn1. 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. However, results from this work show 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 link between RNase MRP and 5.8S 5′ end formation involves RNase MRP cleavage at unknown sites elsewhere in pre-rRNA or in RNA molecules other than pre-rRNA.

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