Erroneous ribosomal RNAs promote the generation of antisense ribosomal siRNA
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA454449 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7088700.fastq.gz | Fastqsanger.gz | |||
| SRR7088695.fastq.gz | Fastqsanger.gz | |||
| SRR7088696.fastq.gz | Fastqsanger.gz | |||
| SRR7088697.fastq.gz | Fastqsanger.gz | |||
| SRR7088698.fastq.gz | Fastqsanger.gz |
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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:Ribosome biogenesis is a multistep process, during which mistakes can occur at any step of pre-rRNA processing, modification, and ribosome assembly. Misprocessed rRNAs are usually detected and degraded by surveillance machineries. Recently, we identified a class of antisense ribosomal siRNAs (risiRNAs) that down-regulate pre-rRNAs through the nuclear RNAi pathway. To further understand the biological roles of risiRNAs, we conducted both forward and reverse genetic screens to search for more suppressor of siRNA (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 the 26S rRNA. C27F2.4(ceBUD23) is an m7G-methyltransferase of the 18S rRNA. E02H1.1(ceDIMT1L) is a predicted m6(2)Am6(2)A-methyltransferase of the 18S rRNA. Mutation of these genes led to a deficiency in modification of rRNAs and elicited accumulation of risiRNAs, which further triggered the cytoplasmic-to-nuclear and cytoplasmic-to-nucleolar translocations of the Argonaute protein NRDE-3. The rRNA processing deficiency also resulted in accumulation of risiRNAs. We also isolated SUSI-3(RIOK-1), which is similar to human RIOK1, that cleaves the 20S rRNA to 18S. We further utilized RNAi and CRISPR-Cas9 technologies to perform candidate-based reverse genetic screens 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 homeostasis of rRNAs.
| S-EPMC6176571 | biostudies-literature
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
Project description:SINEUPs are antisense long non-coding RNAs (lncRNAs) that increase translation of overlapping mRNAs through the activity of two domains: an embedded SINEB2 sequence UP-regulates translation (Effector Domain) while the antisense region provides target specificity (Binding Domain). In this study, we show that the invSINEB2 sequence from the natural SINEUP AS Uchl1 RNA exhibits the functions of an Internal Ribosomal Entry Site (IRES), while viral and cellular IRES sequences act as Effector Domains, promoting protein expression in trans. We demonstrate that the Stem Loop1 of AS Uchl1 SINEB2 is functionally equivalent to the IIId loop of Hepatitis C Virus IRES. The natural circ5533, a circular RNA at the c-myc locus, up-regulates the protein expression in trans of specific target mRNAs through an IRES sequence serving as Effector Domain and antisense regions as Binding Domains. This work shows a new function for embedded transposable elements in lncRNAs, proves that an IRES sequence can work in trans enhancing the translation of a target mRNA and identify a new class of regulatory circRNAs.
2025-08-25 | PXD028571 | Pride
Project description:Background: Eukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs). Methods/Results: We used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPolII or bound to Argonaute (AGO1 by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with PolII occupancy with an average density of 130 (relative units); this decreased to 65%/20 in middle exons and 54%/12 in the last exon. CLASH reads mapping to multi-exon genes showed little distribution bias with an average of about 5 CLASH reads overlapping with 60% of the endo-siRNA reads. However, endo-siRNAs (21-25 nt) intersecting with CLASH reads were enriched at the 5'end and decreased towards the 3'end. We then investigated the 378 genes with particular focus on features indicative for short RNA production; however, found that endo-siRNA numbers did not correlate with gene structures that favor convergent transcription. In contrast, our gene set was found notably over-represented in the NATsDB sense/antisense group as compared to non-overlapping and non-bidirectional groups. Moreover, read counts showed no correlation with the steady-state levels of the related mRNAs and the pattern of endo-siRNAs proved reproducible after an induced mutagenic insult. Conclusions: Our results suggest that antisense transcripts contribute to low levels of endo-siRNAs in fully differentiated human cells. A characteristic endo-siRNA footprint is being produced at sites of RNAPolII transcription which is also related to AGO1. This endo-siRNA signature represents an intriguing finding and its reproducibility suggests that the production of endo-siRNAs is a regulated process with potential homoeostatic impact.
2014-02-28 | GSE52996 | GEO
Project description:Antisense ribosomal siRNAs inhibit RNA polymerase I-directed transcription in C. elegans
| PRJNA693200 | ENA
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:Cis-encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of the vast majority of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content, and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing 9 different asRNAs in M. pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.
2018-10-25 | PXD003231 | Pride
Project description:Background: Eukaryotic cells express a complex layer of noncoding RNAs. An intriguing family of regulatory RNAs includes transcripts from the opposite strand of protein coding genes, so called natural antisense transcripts (NATs). Here, we test the hypothesis that antisense transcription triggers RNA interference and gives rise to endogenous short RNAs (endo-siRNAs). Methods/Results: We used cloned human embryonic kidney cells (HEK293) followed by short RNAseq to investigate the small genic RNA transcriptome. 378 genes gave rise to short RNA reads that mapped to exons of RefSeq genes. The length profile of short RNAs showed a broad peak of 20-24 nucleotides, indicative of endo-siRNAs. Collapsed reads mapped predominantly to the first and the last exon of genes (74%). RNAs reads were intersected with sequences occupied by RNAPolII or bound to Argonaute (AGO1 by crosslinking, ligation, and sequencing of hybrids, CLASH). In the first exon, 94% of the reads correlated with PolII occupancy with an average density of 130 (relative units); this decreased to 65%/20 in middle exons and 54%/12 in the last exon. CLASH reads mapping to multi-exon genes showed little distribution bias with an average of about 5 CLASH reads overlapping with 60% of the endo-siRNA reads. However, endo-siRNAs (21-25 nt) intersecting with CLASH reads were enriched at the 5'end and decreased towards the 3'end. We then investigated the 378 genes with particular focus on features indicative for short RNA production; however, found that endo-siRNA numbers did not correlate with gene structures that favor convergent transcription. In contrast, our gene set was found notably over-represented in the NATsDB sense/antisense group as compared to non-overlapping and non-bidirectional groups. Moreover, read counts showed no correlation with the steady-state levels of the related mRNAs and the pattern of endo-siRNAs proved reproducible after an induced mutagenic insult. Conclusions: Our results suggest that antisense transcripts contribute to low levels of endo-siRNAs in fully differentiated human cells. A characteristic endo-siRNA footprint is being produced at sites of RNAPolII transcription which is also related to AGO1. This endo-siRNA signature represents an intriguing finding and its reproducibility suggests that the production of endo-siRNAs is a regulated process with potential homoeostatic impact. Size selected RNASeq of 3 human embryonic kidney cell (HEK293) samples. 1 control and 2 samples exposed to 100 μg/ml ethyl methanesulfonate for 24 hrs.
2014-02-28 | E-GEOD-52996 | biostudies-arrayexpress
Project description:Transcriptional profiling of HT29 colon cancer cells comparing siRNA-targeted knockdowns of glutaminase (GLUT), cytokeratin 20 (KER), DEAD/H box polypeptide 32 (DEAD), ribosomal protein L32 (RPL32) and transcription factor DP1 (TFDP1) versus scrambled siRNA control and GFP siRNA control Keywords: two-color spotted cDNA microarray, siRNA-targeted gene knockdowns
2008-06-21 | GSE11848 | GEO