Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE165078 | GEO | 2021/01/20
REPOSITORIES: GEO
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Project description:Ribosome biogenesis is a multi-step process, during which mistakes could take place at any step of pre-rRNA processing, modification, and assembly of ribosomes. Misprocessed rRNAs are usually detected and degraded by surveillance machineries. Recently, we identified a class of antisense ribosomal siRNAs (risiRNAs) that downregulate pre-rRNAs through the nuclear RNAi pathway. To further understand the biological roles of risiRNA, we conducted both forward and reverse genetic screenings to search for more susi mutants. We isolated a number of genes that are broadly conserved from yeast to humans and are involved in pre-rRNA modification and processing. Among them, SUSI-2(ceRRP8) is homologous to human RRP8 and engages in m1A methylation of 26S rRNA. C27F2.4(ceBUD23) is a m7G methyltransferase of 18S rRNA. E02H1.1(ceDIMT1L) is a predicted m6(2)Am6(2)A methyltransferase of 18S rRNA . Mutation of these genes led to modification deficiency of rRNAs and elicited accumulation of risiRNAs, which further triggered the cytoplasm to nuclear and nucleolar translocation of an Argonaute protein NRDE-3. The processing deficiency of rRNAs resulted in the accumulation of risiRNAs as well. We isolated SUSI-3(RIOK-1) which is similar to human RIOK1 that cleaves 20S to 18S rRNA. We further utilized RNAi and CRISPR/Cas9 technologies to perform candidate-based reverse genetic screening and identified additional pre-rRNA processing factors that suppressed risiRNA production. Therefore, we concluded that erroneous rRNAs can trigger risiRNA generation and subsequently turn on the nuclear RNAi-mediated gene silencing pathway to inhibit pre-rRNA expression, which may provide a quality control mechanism to maintain the homeostasis of rRNAs.
2018-05-02 | GSE113905 | GEO
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
Project description:Antisense ribosomal siRNAs (risiRNAs) downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3'-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3' to 5' exoribonuclease SUSI-1(ceDIS3L2). There are three polyuridylation polymerases, CDE-1, PUP-2, and PUP-3, in C. elegans. Here, we found that CDE-1 is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates both Argonaute-associated 22G-RNAs and 26S rRNAs at the 3'-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with those results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi. Therefore, this work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.
2020-08-14 | GSE139530 | GEO
Project description:dsRNA-mediated gene silencing (RNAi) can be inherited for multiple generations in C. elegans. To understand this process we conducted a genetic screen for animals defective for transmitting RNAi silencing signals to future generations. This screen identified the gene heritable RNAi defective (hrde)-1. hrde-1 encodes an Argonaute (Ago) that associates with siRNAs in germ cells of the progeny of animals exposed to dsRNA. In nuclei of these germ cells, HRDE-1 engages the Nrde nuclear RNAi pathway to promote RNAi inheritance, and directs H3K9me3 chromatin marks at RNAi targeted genomic loci. Under normal growth conditions, HRDE-1 associates with endogenously expressed small RNAs, which direct nuclear gene silencing in germ cells. In RNAi inheritance mutant animals, silencing is lost over generational time. Concurrently, RNAi inheritance mutant animals exhibit progressively worsening germline defects that ultimately lead to sterility. These results establish that the Ago HRDE-1 directs gene-silencing events in germ cell nuclei, which are required for multi-generational RNAi inheritance and immortality of the germ cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to ensure immortality of the germ cell lineage. H3K9me3 Chip-IP for C. elegans strains nrde-3, nrde-4, glp-4. Small RNA-seq for small RNAs co-immunoprecipitated with HRDE-1
2012-07-30 | E-GEOD-38041 | biostudies-arrayexpress
Project description:Background: Modification of RNAs, particularly at the terminals, is critical for various essential cell processes; for example, uridylation is implicated in tumorigenesis, proliferation, stem cell maintenance, and immune defense against viruses and retrotransposons. Ribosomal RNAs can be regulated by antisense ribosomal siRNAs (risiRNAs), which downregulate pre-rRNAs through the nuclear RNAi pathway in Caenorhabditis elegans. However, the biogenesis and regulation of risiRNAs remain obscure. Previously, we showed that 26S rRNAs are uridylated at the 3'-ends by an unknown terminal polyuridylation polymerase before the rRNAs are degraded by a 3’ to 5’ exoribonuclease SUSI-1(ceDIS3L2).Results: Here, we found that CDE-1, one of the three C.elegans polyuridylation polymerases (PUPs), is specifically involved in suppressing risiRNA production. CDE-1 localizes to perinuclear granules in the germline and uridylates Argonaute-associated 22G-RNAs, 26S and 5.8S rRNAs at the 3’-ends. Immunoprecipitation followed by mass spectrometry (IP-MS) revealed that CDE-1 interacts with SUSI-1(ceDIS3L2). Consistent with these results, both CDE-1 and SUSI-1(ceDIS3L2) are required for the inheritance of RNAi.Conclusions: This work identified a rRNA surveillance machinery of rRNAs that couples terminal polyuridylation and degradation.
2020-08-11 | GSE156000 | GEO
Project description:RNA interference (RNAi) is a conserved gene regulation mechanism that utilizes the Argonaute protein and their associated small RNAs to exert regulatory function on complementary transcripts. While the majority of germline-expressed RNAi pathway components reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here we find that the small RNA biogenesis machinery is spatially and temporally organized during embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Further, we observe that many other RNAi factors form foci in embryonic cells distinct from SIMR granules, including the Argonaute protein CSR-1, underscoring a potential role for cytoplasmic concentrates of RNAi factors to promote gene regulation in embryos. Curiously, coincident with the appearance of the “SIMR granules”, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during early embryogenesis and switching later in embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.
2024-07-31 | GSE273239 | GEO
Project description:To examine the roles of the ADAR genes and the ERI-6/7 helicase in endogenous RNAi pathways, we sequenced small RNA of adr-1; adr-2; eri-6 mutants. We also analyzed the transcriptomes of these mutants using total RNAseq and mRNAseq. mRNAseq was done in mutants depleted for the RNAi factors drh-1 and nrde-3, or control RNAi
2020-01-14 | GSE143595 | GEO
Project description:Gene expression analyses of three-week old soil grown wild-type (Col-3) and apum23-1 mutant. Expression profiling was conducted as an attempt to identify the genes differentially regulated in apum23-1 mutant This study was under taken to characterize a member of pumilio family, APUM23 in Arabidopsis. Pumilio, an RNA-binding protein (RBP) containing tandem repeat PUF domains, has been known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their roles in plants are unknown. We detail the characterization of an Arabidopsis Pumilio gene, APUM23. APUM23 is constitutively expressed with higher level in metabolically active tissues, and upregulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth with serrated and scrunched leaves, abnormal venation pattern, and distorted organization of the palisade parenchyma cells, a phenotype reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on the localization, rRNA processing was examined in apum23 mutant. In apum23, 35S pre-rRNA and unprocessed 18S and 5.8S poly(A) rRNAs were accumulated without affecting steady-state levels of mature rRNAs, indicating that APUM23 participates in the degradation of rRNA by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 snoRNAs, and accumulated RNAs within nucleolus. Our results suggest that APUM23 plays an important role in plant development via rRNA processing and ribosome biogenesis. To identify the genes affected in apum23-1 as compared to wild-type (Col-3) under normal growth conditions using Arabidopsis ATH1 Genome arrays (Affymetrix). RNA samples from these plants were used to generate biotin labelled (cDNA) probes, which were then hybridized to the microarray. For data set generation, three independent samples were used, three from Col-3 and three from apum23-1 plants grown on soil for 21 days.
2011-03-01 | E-GEOD-23940 | biostudies-arrayexpress
Project description:Gene expression analyses of three-week old soil grown wild-type (Col-3) and apum23-1 mutant. Expression profiling was conducted as an attempt to identify the genes differentially regulated in apum23-1 mutant This study was under taken to characterize a member of pumilio family, APUM23 in Arabidopsis. Pumilio, an RNA-binding protein (RBP) containing tandem repeat PUF domains, has been known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their roles in plants are unknown. We detail the characterization of an Arabidopsis Pumilio gene, APUM23. APUM23 is constitutively expressed with higher level in metabolically active tissues, and upregulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth with serrated and scrunched leaves, abnormal venation pattern, and distorted organization of the palisade parenchyma cells, a phenotype reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on the localization, rRNA processing was examined in apum23 mutant. In apum23, 35S pre-rRNA and unprocessed 18S and 5.8S poly(A) rRNAs were accumulated without affecting steady-state levels of mature rRNAs, indicating that APUM23 participates in the degradation of rRNA by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 snoRNAs, and accumulated RNAs within nucleolus. Our results suggest that APUM23 plays an important role in plant development via rRNA processing and ribosome biogenesis.
2011-03-01 | GSE23940 | GEO
Project description:Small RNA pathways play diverse regulatory roles in the nematode C. elegans. However, our understanding of small RNA pathways, their conservation, and their roles in other nematodes is limited. Here, we analyzed small RNA pathways in the parasitic nematode Ascaris. Ascaris has ten Argonautes with five worm-specific Argonautes (WAGOs) that are associated with secondary 5’-triphosphate small RNAs (22-24G-RNAs). These Ascaris WAGOs and their small RNAs target repetitive sequences (WAGO-1, WAGO-2, WAGO-3, and NRDE-3) or mature mRNAs (CSR-1, NRDE-3, and WAGO-3) and are similar to the C. elegans mutator, nuclear, and CSR-1 small RNA pathways. Ascaris CSR-1 likely functions to “license” gene expression in the absence of an Ascaris piRNA pathway. Ascaris ALG-4 and its associated 26G-RNAs target and appear to repress specific mRNAs during meiosis in the testes. Notably, Ascaris WAGOs (WAGO-3 and NRDE-3) small RNAs change their targets between repetitive sequences and mRNAs during spermatogenesis or in early embryos illustrating target plasticity of these WAGOs. We provide a unique and comprehensive view of mRNA and small RNA expression throughout nematode spermatogenesis that illustrates the dynamics and flexibility of small RNA pathways. Overall, our study provides key insights into the conservation and divergence of nematode small RNA pathways.
2021-12-15 | GSE189061 | GEO