Genome-wide chromatin localization maps of the endogenously expressed AID-labelled kinase/ATPase Rio1 in Saccharomyces cerevisiae W303, as determined under unperturbed culture conditions (YPD medium, exponential growth, 23°C)
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE70232 | GEO | 2018/06/14
REPOSITORIES: GEO
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Project description:The conserved Saccharomyces cerevisiae kinase/ATPase Rio1 downregulates rDNA transcription to promote rDNA stability and segregation. To uncover additional roles in transcriptional regulation beyond the rDNA locus we defined the global Rio1 transcriptiome. By NGS we identify 818 differentially expressed genes that are under the transcriptional control of Rio1.
2018-06-14 | GSE70286 | GEO
Project description:In Saccharomyces cerevisiae, the kinase Rio1 regulates rDNA transcription and segregation, pre-rRNA cleavage, and 40S ribosomal subunit maturation. Other roles are unknown. Human orthologue RIOK1; which is frequently overexpressed in malignancies, drives tumor growth and metastasis. Again, also RIOK1 biology is poorly understood. In this study, we charted the global activity of Rio1 in budding yeast. By producing and systems-integrating its protein-interaction, gene-transcription, and chromatin-binding maps we generated Rio1's multi-layered activity network, which controls protein synthesis and turnover, metabolism, growth, proliferation, and genetic stability. Rio1 regulates itself at the transcriptional level, and manages its network both directly and indirectly, via a battery of regulators and transcription factors, including Gcn4. We experimentally confirmed the network and show that Rio1 commands its downstream circuit depending on the growth conditions encountered. We also find that Rio1 and RIOK1 activities are functionally equivalent. Our data suggest that pathological RIOK1 expression may deregulate its network and fuel promiscuous transcription and ribosome production, uncontrolled metabolism, growth, proliferation, and chromosomal instability; well-known contributors to cancer initiation, maintenance and metastasis.
2020-11-23 | GSE107318 | GEO
Project description:Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of noncoding centromere transcripts (cenRNAs). The latter are ensured by downregulation of RNA polymerase II (RNAPII) and turnover by the nuclear exosome. Using S. cerevisiae, we now add kinase Rio1 to this scheme. Yeast cenRNAs are produced in very low amounts either as short (median lengths of 231nt) or long (4,458nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII access and transcription activity, and promotes their turnover by the 5’-3’exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs, which also exist as short transcripts (225nt) at a magnitude higher level than the cenRNAs. In yeast depleted of Rio1, cen- and pericenRNAs accumulatre, and kinetochores misform causing chromosomal instability. The latter phenotypes were also observed with human cells lacking orthologue RioK1, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.
2023-04-20 | GSE218602 | 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:Kinetochores assemble on centromeres via histone H3 variant CENP-A and low levels of noncoding centromere transcripts (cenRNAs). The latter are ensured by transcription factor Cbf1 (S. cerevisiae), centromere-binding protein CENP-B (humans), and the local histone code downregulating RNA polymerase II (RNAPII) activity. CenRNAs levels are further adjusted by the nuclear exosome. Using S. cerevisiae, we now add kinase Rio1 to this scheme. Yeast cenRNAs are produced mostly from the periCEN regions as short (median lengths of 231nt) or long (4,458nt) transcripts, in a 1:1 ratio. Rio1 limits their production by reducing RNAPII access and transcription activity, and promotes their turnover by the 5’-3’exoribonuclease Rat1. Rio1 similarly curtails the concentrations of noncoding pericenRNAs, which nevertheless exist at a magnitude higher level than the cenRNAs. Similar to yeast, human cells depleted of orthologue RioK1 exhibit cenRNA buildup, kinetochore malformation, and chromosomal instability, suggesting that CEN regulation by Rio1/RioK1 is evolutionary conserved.
2023-07-20 | PXD038337 | Pride
Project description:To sustain growth, budding yeast actively transcribes its ribosomal gene array (rDNA) in the nucoleolus to produce ribosomes and proteins. However, intense transcription during rDNA replication may provoke collisions between RNA polymerase I (Pol I) and the replisome, may cause replication fork instability, double-strand breaks, local recombinations and rDNA instability. The latter is manifested by rDNA array expansion or reduction and the formation of extrachromosomal rDNA circles, anomalies that accelerate aging in yeast. Transcription also interferes with the resolution, condensation and segregation of the sister chromatid rDNA arrays. As a consequence, rDNA segregation lags behind the rest of the yeast genome and occurs in late anaphase when rDNA transcription is temporarily shut off. How yeast promotes the stability and transmission of its rDNA array while satisfying a constant need for ribosomes remains unclear. Here we show that the downregulation of Pol I by the conserved cell cycle kinase Rio1 spatiotemporally coordinates rDNA transcription, replication and segregation. More specifically, Rio1 activity promotes copy-number stability of the replicating rDNA array by curtailing Pol I activity and by localising the histone deacetylase Sir2, which establishes a heterochromatic state that silences rDNA transcription. At anaphase entry, Rio1 and the Cdc14 phosphatase target Pol I subunit Rpa43 to dissociate Pol I from the 35S rDNA promoter. The rDNA locus then condensates and segregates, thereby concluding the genome transmission process. Rio1 is involved in ribosome maturation in the cytoplasm of budding yeast and human cells. Additional engagements in the cytoplasm or roles in the nucleus are unknown. Our study describes its first nuclear engagement as a Pol I silencing kinase. This activity may prove highly relevant as dysregulated RNA polymerase I activity has been associated with cancer initiation and proliferation.
2017-02-10 | GSE54930 | GEO
Project description:In this study, we performed quantitative phosphoproteomic analysis on S. islandicus REY15A either treated with UV or in the absence of Rio1.
2020-11-05 | PXD021663 | Pride
Project description:The global map of Rio1 activity in Saccharomyces cerevisiae
| PRJNA419752 | ENA
Project description:Identification of in vivo direct RNA targets for RNA binding proteins (RBP) provides critical insight into their regulatory activities and mechanisms. Recently, we described a methodology for enhanced crosslinking and immunoprecipitation followed by high-throughput sequencing (eCLIP-seq) using antibodies against endogenous RNA binding proteins. However, in many cases it is desirable to profile targets of an RNA binding protein for which an immunoprecipitation-grade antibody is lacking. Here we describe a scalable method for using CRISPR/Cas9-mediated homologous recombination to insert a peptide tag into the endogenous RNA binding protein locus. Further, we show that TAG-eCLIP performed using tag-specific antibodies can yield the same robust binding profiles after proper control normalization as eCLIP with antibodies against endogenous proteins. Finally, we note that antibodies against commonly used tags can immunoprecipitate significant amounts of antibody-specific RNA, emphasizing the need for paired controls alongside each experiment for normalization. TAG-eCLIP enables eCLIP profiling of new native proteins where no suitable antibody exists, expanding the RBP-RNA landscape.
2017-08-22 | GSE88722 | GEO
Project description:Rio1 downregulates centromeric RNA levels to promote the timely assembly of structurally fit kinetochores
| PRJNA904388 | ENA