Regulation of translation by site-specific ribosomal RNA methylation [RiboMeth-Seq]
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE153473 | GEO | 2021/08/16
REPOSITORIES: GEO
Similar Datasets
Project description:We report that 2’-O-methylation levels at subset of positions in human ribosomal RNA are variable between cell types and conditions, and that the degree of methylation at distinct sites is responsive to key cellular pathways. MYC overexpression results in increased methylation at a particular rRNA site (18S:C174). We find that this methylation is important for modulating translation of distinct mRNAs, leading to phenotypic changes including modulation of cell proliferation rate.
2021-08-16 | GSE148965 | GEO
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:N6-methyladenosine (m6A) is a conserved nucleoside modification that regulates many facets of RNA metabolism and cellular physiology. Using quantitative mass spectrometry, we find that the universally conserved tandem adenosines at the 3’ end of 18S rRNA (A1781 and A1782 in Saccharomyces cerevisiae), which were thought to be constitutively di-methylated (i.e. N6, N6-dimethyladenosine or m62A), are also mono-methylated (i.e. m6A). Although present at substoichiometric amounts, m6A at these positions increases significantly and specifically in response to sulfur starvation in both yeast and mammalian cells. Combining yeast genetics and ribosome-profiling, we further demonstrate that ribosomes bearing different numbers of methyl groups (zero, one, and two) at these tandem adenosines exhibit distinct translation profiles in a sulfur-dependent fashion. Our work thus reveals methylation multiplicity as a mechanism to regulate translation and also uncovers the functional importance of the ubiquitous m62A modification in 18S rRNA.
2021-03-11 | GSE142528 | GEO
Project description:The 18S rRNA sequence is highly conserved, particularly at its 3’-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3’-end is degenerate with similar sites nearby. Here we used yeast genetics, biochemistry, and next generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3’-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control step, but not in healthy cells with intact quality control mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA quality control. Altogether, the data support a model in which Rio1 inspects the 3’-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.
2024-03-03 | GSE259239 | GEO
Project description:The major information-carrying macromolecules in the cell, DNA, RNA, and protein, carry an additional layer of information on top of their sequence in the form of modifications of residues. The modifications provide additional functional groups and impact the structure and function of the molecules. Cellular RNA molecules contain more than 100 different modifications and are found in all domains of life and in all major classes of RNA in eukaryotic organisms. Together these modifications constitute the epitranscriptome of which two-thirds are methylations with 2’-O methylation of the ribose moiety of the nucleotide as the most abundant. Many aspects of ribose methylation are underexplored because the existing methods for their detection are laborious and can only address a few modification sites at a time. Here, we introduce RiboMeth-Seq, a high-throughput sequencing based method and applies it to yeast ribosomal RNA. We detect all of the known as well as one new methylation site and provide evidence for hypomethylation at specific residues. Furthermore we demonstrate that many methylation events are interdependent and outline the timing of modifications during ribosome biogenesis. Our results demonstrate a novel and efficient approach to understanding of the role of modifications in ribosomal RNA folding and ribosome function. Recent evidence point to changes in ribose methylation patterns in cancer ribosomes and we anticipate that RiboMeth-Seq can be applied here as well as to other diseases in which ribosomes are affected, including the heritable ribosomopathies. Yeast RNA was degraded at denaturing conditions into small fragments, long enough to be mapped by sequence alignment to the reference genome (20-40 nucleotides). The fragments were ligated to RNA oligos using a tRNA ligase that was mutated to remove its kinase activity, reverse transcribed and the cDNA used as input for Ion semiconductor sequencing. The first and last nucleotides of the inserts were recorded using the full sequence for mapping and the read-ends plotted against the sequence. 2'-O-Methylated RNA positions are protected from cleavage, and read ends from such positions are therefore underrepresented compared to the surrounding positions.
2014-11-24 | E-GEOD-54655 | biostudies-arrayexpress
Project description:Non-coding RNAs including small nucleolar RNAs (snoRNAs) play important roles in leukemogenesis but the relevant mechanisms remain incompletely understood. We performed snoRNA focused CRISPR-Cas9 knockout library screenings which targeted the entire snoRNAnome and corresponding host genes. The C/D box containing SNORD42A was identified as an essential modulator for AML cell survival and proliferation in multiple human leukemia cell lines. In line, SNORD42A was consistently expressed at high levels in primary AML patient samples. Functionally, knockout of SNORD42A reduced colony formation capability and inhibited proliferation. The SNORD42A acts as a C/D box snoRNA and directs 2´-O-methylation at Uridine 116 of 18S rRNA. Deletion of SNORD42A decreased 18S-U116 2´-O-methylation which was associated with a specific decrease in the translation of ribosomal proteins. In line, the cell size of SNORD42A deletion carrying leukemia cells was decreased. Taken together, these findings establish that high level expression of SNORD42A with concomitant U116 18S rRNA 2´-O-methylation is essential for leukemia cell growth and survival.
2020-02-25 | GSE140355 | GEO
Project description:Purpose: Pre-ribosomal RNA is cleaved at defined sites, but many endonucleases involved in 18S rRNA release are not known. We apply an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between a yeast candidate pre-rRNA endonuclease (Utp24) and its targets in a living cell. Methods: We apply CRAC to an HTP-tagged Utp24 protein (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A). At least two independent experiments were performed and analyzed separately. Results: We found that yeast Utp24 UV-crosslinked in vivo to the U3 snoRNA and all (pre-)rRNA elements that form the central pseudoknot in the 18S rRNA. The pseudoknot is an evolutionarily highly conserved structure that is required to ensure pre-rRNA processing at three cleavage sites (A0, A1 and A2) and still present in the mature rRNA. According to our crosslinking data, the endonuclease Utp24 is placed in close proximity to site A1 at the 5'-end of the 18S rRNA. Conclusion: Our study strongly supports the hypothesis that Utp24 cleaves pre-rRNA at sites A1 and A2. Examination of targets for pre-rRNA endonucleases in yeast cells.
2016-04-01 | E-GEOD-75991 | biostudies-arrayexpress
Project description:In eukaryotes, biogenesis of ribosomes requires folding and assembly of the precursor rRNA (pre-rRNA) with a large number of proteins and snoRNPs into huge RNA-protein complexes. In spite of intense genetic, biochemical and high resolution cryo-EM studies in Saccharomyces cerevisiae, information about the conformation of the earliest 35S pre-rRNA is limited. To overcome this, we performed high-throughput SHAPE chemical probing on the 35S pre-rRNA associated with 90S pre-ribosomes. We focused our analyses on external (5´ETS) and internal (ITS1) transcribed spacers as well as the 18S region. We show that in the 35S pre-rRNA, the central region of the 18S is in a more open configuration compared to 20S pre-rRNA and that the central pseudoknot is not formed. The essential ribosome biogenesis protein Mrd1 influences the structure of the 18S part locally and is involved in organizing the central pseudoknot and surrounding structures. Our results demonstrate that the U3 snoRNA dynamically interacts with the 35S pre-rRNA and that Mrd1 is required for disrupting U3 snoRNA base-pairing interactions in the 5'ETS. We propose that the dynamic U3 snoRNA interactions and Mrd1 are essential for establishing the structure of the central region of 18S that is required for processing and 40S subunit function.
2018-01-15 | GSE106868 | GEO
Project description:Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to down-regulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing, and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an unexpected explanation for the cytotoxic effects of loss of CpG methylation. Agilent custom microarray analyses of transcripts from sense and antisense strands of the human rRNA genes present in total nuclear RNA from HCT116-DNMT1-/-;DNMT3b-/- (DKO) and HCT116 (PS) cells. The DKO/PS RNA ratio was normalized within the 5â region of the 45S ribosomal RNA, since this sense coding region was found by quantitative Northern and S1-mapping analyses to equally present in the RNA pools from both cells (as compared to total RNA levels and 18S and 28S levels). The distribution for sense RNAs from the coding region is strongly influenced by the predominant RPI 45S rRNA transcript and hence peaks at 0, the DKO and PS 45S rRNA signals having been normalized. 60-mer probes were designed to tile Human rDNA (GenBank accession U13369) on both strands using ArrayOligoSelector (Wardle et al., 2006). A total of 1,146 different non-overlapping probes (573 for each DNA strand) were designed to cover the 43 kb of human rDNA and each printed in triplicate on the array. The experiment was performed using a dye-swap duplicate analysis. The microarrays were scanned on a G2565CA DNA microarray scanner (Agilent Technologies) and the quantification performed using the Feature Extraction software (Agilent technologies). The data were analyzed using the LIMMA software package in R (Smyth and Speed, 2003).
2010-05-16 | E-GEOD-17042 | biostudies-arrayexpress
Project description:Epigenetic methyl-CpG silencing of the ribosomal RNA (rRNA) genes is thought to down-regulate rRNA synthesis in mammals. In contrast, we now show that CpG methylation in fact positively influences rRNA synthesis and processing. Human HCT116 cells, inactivated for DNMT1 and DNMT3b or treated with aza-dC, lack CpG methylation and reactivate a large fraction of normally silent rRNA genes. Unexpectedly, these cells display reduced rRNA synthesis and processing, and accumulate unprocessed 45S rRNA. Reactivation of the rRNA genes is associated with their cryptic transcription by RNA polymerase II. Ectopic expression of cryptic rRNA gene transcripts recapitulates the defects associated with loss of CpG methylation. The data demonstrate that rRNA gene silencing prevents cryptic RNA polymerase II transcription of these genes. Lack of silencing leads to the partial disruption of rRNA synthesis and rRNA processing, providing an unexpected explanation for the cytotoxic effects of loss of CpG methylation. Agilent custom microarray analyses of transcripts from sense and antisense strands of the human rRNA genes present in total nuclear RNA from HCT116-DNMT1-/-;DNMT3b-/- (DKO) and HCT116 (PS) cells. The DKO/PS RNA ratio was normalized within the 5’ region of the 45S ribosomal RNA, since this sense coding region was found by quantitative Northern and S1-mapping analyses to equally present in the RNA pools from both cells (as compared to total RNA levels and 18S and 28S levels). The distribution for sense RNAs from the coding region is strongly influenced by the predominant RPI 45S rRNA transcript and hence peaks at 0, the DKO and PS 45S rRNA signals having been normalized.
2009-07-11 | GSE17042 | GEO