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Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans.

ABSTRACT: Eukaryotic 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 the association of NRDE proteins with pre-rRNAs and the silencing of pre-rRNAs. In the presence of risiRNAs, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNAs inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the RNAi-targeted site. Meanwhile, exosomes mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results established a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.

SUBMITTER: Liao S

PROVIDER: S-EPMC8450093 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

Project description:Eukaryotic 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 risiRNAs, NRDE-2 accumulated in the nucleolus and colocalized with RNA polymerase I. risiRNAs inhibited the transcription elongation of RNA polymerase I by decreasing RNAP I occupancy downstream of the RNAi-targeted site. Meanwhile, exosomes mislocalized from the nucleolus to nucleoplasm in suppressor of siRNA (susi) mutants, in which erroneous rRNAs accumulated. These results established a novel model of rRNA surveillance by combining ribonuclease-mediated RNA degradation with small RNA-directed nucleolar RNAi system.

2021-01-20 | GSE165078 | GEO

Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

Project description:Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans

| PRJNA693200 | ENA

BLM helicase facilitates RNA polymerase I-mediated ribosomal RNA transcription.

Project description:Bloom's syndrome (BS) is an autosomal recessive disorder that is invariably characterized by severe growth retardation and cancer predisposition. The Bloom's syndrome helicase (BLM), mutations of which lead to BS, localizes to promyelocytic leukemia protein bodies and to the nucleolus of the cell, the site of RNA polymerase I-mediated ribosomal RNA (rRNA) transcription. rRNA transcription is fundamental for ribosome biogenesis and therefore protein synthesis, cellular growth and proliferation; its inhibition limits cellular growth and proliferation as well as bodily growth. We report that nucleolar BLM facilitates RNA polymerase I-mediated rRNA transcription. Immunofluorescence studies demonstrate the dependance of BLM nucleolar localization upon ongoing RNA polymerase I-mediated rRNA transcription. In vivo protein co-immunoprecipitation demonstrates that BLM interacts with RPA194, a subunit of RNA polymerase I. (3)H-uridine pulse-chase assays demonstrate that BLM expression is required for efficient rRNA transcription. In vitro helicase assays demonstrate that BLM unwinds GC-rich rDNA-like substrates that form in the nucleolus and normally inhibit progression of the RNA polymerase I transcription complex. These studies suggest that nucleolar BLM modulates rDNA structures in association with RNA polymerase I to facilitate RNA polymerase I-mediated rRNA transcription. Given the intricate relationship between rDNA metabolism and growth, our data may help in understanding the etiology of proportional dwarfism in BS.

| S-EPMC3277314 | biostudies-literature

DNA-directed termination of RNA polymerase II transcription.

Project description:RNA polymerase II (RNAPII) transcription involves initiation from a promoter, transcriptional elongation through the gene, and termination in the terminator region. In bacteria, terminators often contain specific DNA elements provoking polymerase dissociation, but RNAPII transcription termination is thought to be driven entirely by protein co-factors. We used biochemical reconstitution, single-molecule studies, and genome-wide analysis in yeast to study RNAPII termination. Transcription into natural terminators by pure RNAPII results in spontaneous termination at specific sequences containing T-tracts. Single-molecule analysis indicates that termination involves pausing without backtracking. The "torpedo" Rat1-Rai1 exonuclease (XRN2 in humans) greatly stimulates spontaneous termination but is ineffectual on other paused RNAPIIs. By contrast, elongation factor Spt4-Spt5 (DSIF) suppresses termination. Genome-wide analysis further indicates that termination occurs by transcript cleavage at the poly(A) site exposing a new 5' RNA-end that allows Rat1-Rai1 loading, which then catches up with destabilized RNAPII at specific termination sites to end transcription.

| S-EPMC7615648 | biostudies-literature

A Caenorhabditis elegans RNA-directed RNA polymerase in sperm development and endogenous RNA interference.

Project description:Short interfering RNAs (siRNAs) are a class of regulatory effectors that enforce gene silencing through formation of RNA duplexes. Although progress has been made in identifying the capabilities of siRNAs in silencing foreign RNA and transposable elements, siRNA functions in endogenous gene regulation have remained mysterious. In certain organisms, siRNA biosynthesis involves novel enzymes that act as RNA-directed RNA polymerases (RdRPs). Here we analyze the function of a Caenorhabditis elegans RdRP, RRF-3, during spermatogenesis. We found that loss of RRF-3 function resulted in pleiotropic defects in sperm development and that sperm defects led to embryonic lethality. Notably, sperm nuclei in mutants of either rrf-3 or another component of the siRNA pathway, eri-1, were frequently surrounded by ectopic microtubule structures, with spindle abnormalities in a subset of the resulting embryos. Through high-throughput small RNA sequencing, we identified a population of cellular mRNAs from spermatogenic cells that appear to serve as templates for antisense siRNA synthesis. This set of genes includes the majority of genes known to have enriched expression during spermatogenesis, as well as many genes not previously known to be expressed during spermatogenesis. In a subset of these genes, we found that RRF-3 was required for effective siRNA accumulation. These and other data suggest a working model in which a major role of the RRF-3/ERI pathway is to generate siRNAs that set patterns of gene expression through feedback repression of a set of critical targets during spermatogenesis.

| S-EPMC2787422 | biostudies-literature

RNA-polymerase-I-directed rDNA transcription, life and works.

Project description:In the extensive network of interdependent biochemical processes required for cell growth and division, there is mounting evidence that ribosomal DNA transcription by RNA polymerase I (pol I) not only drives cell growth via its direct role in production of the ribosomal RNA (rRNA) component of the protein-synthesis machinery, but that it is also crucial in determining the fate of the cell. Considerable progress has been made in recent years towards understanding both the function of components of the pol I transcription machinery and how cells accomplish the tight control of pol I transcription, balancing the supply of rRNA with demand under different growth conditions.

| S-EPMC3858833 | biostudies-literature

RNA-dependent RNA polymerases-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway in C. elegans

Project description:The biogenesis of rRNA drives cell growth and proliferation, but mechanisms that modulate rRNA production remain poorly understood. We have conducted a genetic screen to look for factors that negatively regulate endo-siRNA generation in C. elegans, and identified the suppressor of siRNA (susi)-1and a new class of triphosphorylated antisense ribosomal siRNAs (risiRNAs). In susi-1 mutant, we observed an accumulation of siRNA sequences complementary to 18S and 26S rRNAs. risiRNAs are similar to 22G-RNA, as they are 22 nt in length and have a triphosphorylated guanidine at their 5'-end. The generation of risiRNAs depends on RNA-dependent RNA polymerases, but not the ERI/Dicer complex. risiRNAs act through the nuclear RNAi pathway and guide the Argonaute protein NRDE-3 to the nucleoli, which leads to a downregulation of pre-rRNA expression. Interestingly, low temperature and ultraviolet irradiation also induces the accumulation of risiRNA, suggesting potential regulatory functions. Cloned SUSI-1 shows homology to Dis3-like exonuclease 2 (Dis3L2) in other organisms, a gene identified as a key exonuclease involved in the 3' to 5' degradation of oligouridylated RNAs. We observed an accumulation of 3'-tail oligouridylated 26S rRNA in susi-1 mutant and in low temperature-treated animals. Consistently, the injection of 3'-tail oligouridylated 26S rRNA elicited a nuclear accumulation of NRDE-3. Our findings thus establish SUSI-1(ceDis3L2) as a suppressor of endo-siRNA generation, identify a novel class of risiRNAs that suppress pre-rRNA expression, and suggest a potential disease mechanism.

2016-12-07 | GSE90927 | GEO

Old drug, new target: ellipticines selectively inhibit RNA polymerase I transcription.

Project description:Transcription by RNA polymerase I (Pol-I) is the main driving force behind ribosome biogenesis, a fundamental cellular process that requires the coordinated transcription of all three nuclear polymerases. Increased Pol-I transcription and the concurrent increase in ribosome biogenesis has been linked to the high rates of proliferation in cancers. The ellipticine family contains a number of potent anticancer therapeutic agents, some having progressed to stage I and II clinical trials; however, the mechanism by which many of the compounds work remains unclear. It has long been thought that inhibition of Top2 is the main reason behind the drugs antiproliferative effects. Here we report that a number of the ellipticines, including 9-hydroxyellipticine, are potent and specific inhibitors of Pol-I transcription, with IC(50) in vitro and in cells in the nanomolar range. Essentially, the drugs did not affect Pol-II and Pol-III transcription, demonstrating a high selectivity. We have shown that Pol-I inhibition occurs by a p53-, ATM/ATR-, and Top2-independent mechanism. We discovered that the drug influences the assembly and stability of preinitiation complexes by targeting the interaction between promoter recognition factor SL1 and the rRNA promoter. Our findings will have an impact on the design and development of novel therapeutic agents specifically targeting ribosome biogenesis.

| S-EPMC3576063 | biostudies-literature

DNA Intercalators Inhibit Eukaryotic Ribosomal RNA Synthesis by Impairing the Initiation of Transcription.

Project description:In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system.

| S-EPMC8466728 | biostudies-literature

Che-1/AATF binds to RNA polymerase I machinery and sustains ribosomal RNA gene transcription.

Project description:Originally identified as an RNA polymerase II interactor, Che-1/AATF (Che-1) has now been recognized as a multifunctional protein involved in cell-cycle regulation and cancer progression, as well as apoptosis inhibition and response to stress. This protein displays a peculiar nucleolar localization and it has recently been implicated in pre-rRNA processing and ribosome biogenesis. Here, we report the identification of a novel function of Che-1 in the regulation of ribosomal RNA (rRNA) synthesis, in both cancer and normal cells. We demonstrate that Che-1 interacts with RNA polymerase I and nucleolar upstream binding factor (UBF) and promotes RNA polymerase I-dependent transcription. Furthermore, this protein binds to the rRNA gene (rDNA) promoter and modulates its epigenetic state by contrasting the recruitment of HDAC1. Che-1 downregulation affects RNA polymerase I and UBF recruitment on rDNA and leads to reducing rDNA promoter activity and 47S pre-rRNA production. Interestingly, Che-1 depletion induces abnormal nucleolar morphology associated with re-distribution of nucleolar proteins. Finally, we show that upon DNA damage Che-1 re-localizes from rDNA to TP53 gene promoter to induce cell-cycle arrest. This previously uncharacterized function of Che-1 confirms the important role of this protein in the regulation of ribosome biogenesis, cellular proliferation and response to stress.

| S-EPMC7293028 | biostudies-literature

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