Compound heterozygous mutations in the noncoding RNU4ATAC gene affect the homeostasis and function of the Integrator complex
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ABSTRACT:
PROVIDER: PRJNA809928 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR18121607_1.fastq.gz | Fastqsanger.gz | |||
| SRR18121607_2.fastq.gz | Fastqsanger.gz | |||
| SRR18121608_1.fastq.gz | Fastqsanger.gz | |||
| SRR18121608_2.fastq.gz | Fastqsanger.gz | |||
| SRR18121609_1.fastq.gz | Fastqsanger.gz |
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Project description:Various genetic diseases associated with microcephaly and developmental defects are due to pathogenic variants in the U4atac snRNA, a component of the minor spliceosome essential for the removal of U12-type introns from eukaryotic messenger RNAs. While it has been shown that a few RNU4ATAC mutations result in impaired binding of essential protein components, the molecular defects of the vast majority are still unknown. Here, we used cells derived from two RNU4ATAC compound heterozygous (g.108_126del;g.111G>A) twin patients with MOPD1 phenotypes to analyze the molecular consequences of the mutations on snRNPs formation and splicing. We show that the U4atac108_126del mutant is unstable and that the 111G>A mutation located in the apical 3’ stem-loop of U4atac induces defects in 3’-end maturation giving rise to decreased levels of mature U4atacG111A snRNA as well as of minor di- and tri-snRNPs. We also found that the mutant cells have alterations in splicing of minor introns and contain lower levels of INTS7 and INTS10 proteins. Altogether, our results demonstrate that the compound heterozygous g.108_126del;g.111G>A U4atac mutations lead to defects in 3’-end processing of U4atac transcripts and affect the homeostasis and function of the Integrator complex.
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Project description:RNA Polymerase II (RNAPII) controls expression of all protein coding genes and most noncoding loci in higher eukaryotes. Calibrating RNAPII activity requires an assortment of polymerase-associated factors that are recruited at sites of active transcription. The Integrator complex is one of the most elusive transcriptional regulators in metazoans, deemed to be recruited after initiation to help establish and modulate paused RNAPII. Integrator is known to be composed of 14 subunits that assemble and operate in a modular fashion. We employed proteomics and machine-learning structure prediction (AlphaFold2) to identify an additional Integrator subunit, INTS15. We report that INTS15 assembles primarily with the INTS13/INTS14/INTS10 module and interfaces with the Int-PP2A module. Functional genomics analysis further reveals a role for INTS15 in modulating RNAPII pausing at a subset of genes. Our study shows that omics approaches combined with AlphaFold2-based predictions provide additional insights into the molecular architecture of large and dynamic multiprotein complexes.
2023-02-10 | PXD039995 | Pride
Project description:1. Evaluate the diagnostic value of long noncoding RNA (CCAT1) expression by RT-PCR in peripheral blood in colorectal cancer patients versus normal healthy control personal.
2. Evaluate the clinical utility of detecting long noncoding RNA (CCAT1) expression in diagnosis of colorectal cancer patients & its relation to tumor staging.
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Project description:RNA polymerase II (RNAPII) controls the expression of all protein coding genes and most noncoding loci in higher eukaryotes. The ultimate rate of transcription is regulated at the level of multiple checkpoints during the transcription cycle (initiation, pausing, termination). Calibrating the activity of RNAPII is fundamental to all cellular processes and requires an assortment of polymerase-associated factors that are recruited at sites of active transcription. The Integrator complex is one of the most elusive transcriptional regulators in metazoans, deemed to be recruited after initiation to help establish and modulate paused RNAPII. Recent structural, biochemical, and functional evidence suggest that Integrator is composed of 14 subunits that assemble and operate in a modular fashion. We employed proteomics and machine-learning structure prediction (AlphaFold2) approaches to identify a novel Integrator subunit, INTS15. We report that INTS15 assembles primarily with the INTS13/INTS14/INTS10 module and interfaces with the core of the Int-PP2A complex. Functional genomics analysis further reveals a role for INTS15 in modulating RNAPII pausing at a subset of genes in human cell lines. Our study shows that omics approaches combined with AlphaFold2-based predictions provide novel insights into the molecular architecture of large and flexible multiprotein complexes.
2023-07-20 | PXD039956 | Pride
Project description:Integrator regulates the 3’-end processing and termination of multiple classes of noncoding RNAs. Depletion of INTS11, the catalytic subunit of Integrator, or ectopic expression of its catalytic dead enzyme impairs the 3’-end processing and termination of a set of protein coding transcripts termed Integrator-regulated termination (IRT) genes. This defect is manifested by increased RNA polymerase II (RNAPII) readthrough and occupancy of serine-2 phosphorylated RNAPII, de-novo trimethylation of lysine 36 on histone H3, and a compensatory elevation of the cleavage and polyadenylation (CPA) complex in the downstream regions. 3’-RNA-sequnecing reveals that proximal polyadenylation site usage relies on the endonuclease activity of INTS11. The DNA sequence encompassing the transcription end sites of IRT genes feature downstream polyadenylation motifs and an enrichment of GC content that permits the formation of secondary structures within the 3’UTR. Taken together, this study identifies a subset of protein-coding transcripts whose 3’-end processing requires the Integrator complex.
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Project description:Alternative 3’-end RNA processing provides an important source of transcriptome diversification, which impacts various biological processes and the etiology of diseases, including cancer. Here, we focused on a transcript isoform switch that leads to the readthrough expression of the long noncoding RNA NEAT1_2, at the expense of the shorter polyadenylated transcript NEAT1_1. Expression of NEAT1_2 is required for the formation of paraspeckles (PS), nuclear bodies that protect cancer cells from oncogene-induced replication stress and chemotherapy. Searching for proteins that interact with endogenous NEAT1 by RNA Affinity Purification coupled to mass spectrometry (RAP-MS), we identified factors involved in the 3’-end processing of polyadenylated (pA+) RNA as well as several components of the Integrator complex, known to process the 3’-ends of RNAs lacking polyadenylation sites. Unexpectedly, genetic perturbation experiments established that Integrator restraints NEAT1_2 expression, and thereby PS assembly, by promoting the 3’-end processing of pA+ NEAT1_1. Consistently, low expression levels of several Integrator subunits correlated with poorer prognosis of cancer patients exposed to chemotherapeutics. Our study identifies Integrator as a key regulator of PS biogenesis and highlights a previously unrecognized ability of the complex to process pA+ RNA. The data also establish a link between Integrator, cancer biology and chemosensitivity, which may be exploited therapeutically.
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Project description:The complete pool of barcoded essential heterozygous diploid deletion strains of S. cerevisiae were screened with 20 compounds from the Chembridge NOVACore chemical library to identify gene deletions that confer sensitivity to each compound.
2011-04-13 | E-TABM-977 | biostudies-arrayexpress
Project description:Mutations in the RYR1 gene are the most common cause of human congenital myopathies and patients with recessive mutations are severely affected and characteristically display ptosis and/or ophthalmoplegia. In order to gain insight into the mechanism leading to extraocular muscle involvement, we investigated the biochemical, structural and physiological properties of eye muscles from mouse models we created knocked-in for RYR1 mutations. Ex vivo force production in extraocular muscles from compound heterozygous RyR1p.Q1970fsX16+p.A4329D mutant mice was significantly reduced compared to that observed in WT. The decrease in muscle force was also accompanied by approximately a 40% reduction in RyR1 protein content, a decrease in electrically evoked calcium transients, disorganization of the muscle ultrastructure and a decrease in the number of calcium release units. Unexpectedly, the superfast and ocular-muscle specific myosin heavy chain-EO isoform was almost undetectable in RyR1p.Q1970fsX16+p.A4329D mutant mice. The results of this study show for the first time that the extraocular muscle phenotype caused by compound heterozygous RYR1 mutations is due to reduced content of ryanodine and dihydropyridine receptors, the presence of fewer calcium release units and associated mitochondria as well as disorganization of myofiber structure. Additionally, the presence of the two mutations leads to the almost complete absence of the extraocular muscle-specific isoform of myosin heavy chain.
2023-03-15 | PXD016879 | Pride
Project description:Complexes containing INTS3 and either NABP1 or NABP2 were initially characterized in DNA damage responses, but their biochemical function remained unknown. Using affinity purifications and HIV Integration Targeting-Sequencing (HIT-Seq), we find that these complexes are part of the Integrator complex, which binds RNA Polymerase II and regulates specific target genes. Integrator cleaves snRNAs as part of their processing to their mature form in a mechanism that is intimately coupled with transcription termination. However, HIT-Seq reveals that Integrator also binds to the 3’ end of replication-dependent histones and promoter proximal regions of genes with polyadenylated transcripts. Depletion of Integrator subunits results in transcription termination failure, disrupting histone mRNA processing, and leading to polyadenylation of snRNAs and histone mRNAs. Furthermore, promoter proximal binding of Integrator negatively regulates expression of genes whose transcripts are normally polyadenylated. Integrator recruitment to all three gene classes is DSIF-dependent, suggesting that Integrator functions as a termination complex at DSIF-dependent RNA Polymerase II pause sites. HITseq was used to interrogate chromatin binding sites of of proteins in the complex (including INIP, INTS3, INTS9, INTS11, NABP1, NABP2, NELFA, NELFB, NELFCD, NELFE, SPT5). These proteins were fused to LEDGF-IBD and expressed in mouse LEDGF-/-MEF cells. The fusion proteins then target HIV integration to the chromatin binding sites. The HIV integration sites were identified using linker-mediated PCR and highthroughput sequencing and serve as surrogate for chromatin binding sites. Mapping was done using mouse genome mm9.
2015-01-20 | E-GEOD-65090 | biostudies-arrayexpress