CAPTURE of the human U2 snRNA genes expands the repertoire of associated factors.
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ABSTRACT:
PROVIDER: PRJNA810044 | ENA |
REPOSITORIES: ENA
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Project description:In order to identify factors involved in transcription of human snRNA genes and 3’ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly-repeated U2 snRNA genes in human cells. CAPTURE enriched many factors expected to be associated with human snRNA genes including pol II, NELF, CDK9, SPT5 and several subunits of the Integrator Complex. SPT6, Mediator subunits 16, 20 and 23, TAF15, CDK7, CDK9, Cyclin K, and the SPT16 subunit of FACT were also enriched. Several polyadenylation factors, including CPSF1, CPSF6 and CstF1-3 were also enriched by U2 gene CAPTURE. These add to the polyadenylation factors CstF2, Pcf11 and Ssu72 which we previously showed are associated with the human U1 and U2 genes. Analysis of genome-wide studies and ChIP-qPCR confirms the association of SPT6 with the U2 genes. In addition, SPT6 knockdown causes loss of subunit 3 of the Integrator complex from the U2 genes and transcriptional readthrough, indicating a functional role in snRNA gene expression. CAPTURE has therefore expanded the repertoire of transcription and RNA processing factors associated with these genes.
2022-05-18 | GSE197372 | GEO
Project description:In order to identify factors involved in transcription of human snRNA genes and 3’ end processing of the transcripts, we have carried out CRISPR affinity purification in situ of regulatory elements (CAPTURE), which is deadCas9-mediated pull-down, of the tandemly-repeated U2 snRNA genes in human cells. Pull-down proteins were identified by mass spectrometry-based proteomics analysis.
2022-06-09 | PXD033580 | Pride
Project description:Spliceosomal snRNA are key components of small nuclear ribonucleoprotein particles (snRNPs), the building blocks of the spliceosome. The biogenesis of snRNPs is a complex process involving multiple cellular and subcellular compartments, the details of which are yet to be described. In short, the snRNA is exported to the cytoplasm as 3‘-end extended precursor (pre-snRNA), where it acquires a heptameric Sm ring. The SMN complex which catalyses this step, recruits Sm proteins and assembles them around the pre-snRNA at the single stranded Sm site. After additional modification, the complex is re-imported into the nucleus where the final maturation step occurs. Our modeling suggests that during the cytoplasmic stage of maturation pre-snRNA assumes a compact secondary structure containing Near Sm site Stem (NSS) which is not compattible with the formation of the Sm ring. To validate our in silico predictions we employed selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) on U2 snRNA in vivo, ex vivo and in vitro, and U4 pre-snRNA in vitro. For the in vivo experiment HeLa cells were incubated for 10 min at 37°C with NAI or DMSO to final concentration 200 mM. RNA was isolated using Trizol (Sigma) and 200 µl chloroform and precipitated with ethanol at -20°C overnight. For the ex vivo experiment, RNA was isolated from HeLa cells after Protease K treatment at room temperature for 45 min. After incubation, RNA was isolated using equilibrated phenol/chloroform/isoamyl alcohol buffered by folding buffer (110 mM HEPES pH 8.0, 110 mM KCl, 11 mM MgCl2) and cleaned on a PD-10 column according to the manufacturer’s instructions. Isolated RNA was treated with 100mM NAI or DMSO for 10 min at 37°C. For the in vitro experiment, U2WT and U4 pre-snRNA were transcribed by T7 polymerase followed by DNase I (30 min at 37 °C) and Proteinase K (30 min at 37°C) treatments. U2 snRNA was purified on 30 kDa Amicon columns, folded for 30 min at 37°C in 57 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. U4 pre-snRNA was purified on Superdex 200 Increase 10/300GL, folded for 30 min at 37°C in 60 mM MgCl2 and incubated with 100 mM NAI at 37°C for 10 min. DMSO was used as a negative control. All prepared RNA samples (in vitro, ex vivo, in vivo) were used for reverse transcription with the gene-specific primer 5’-CGTTCCTGGAGGTACTGCAA for U2 snRNA and 5’- AAAAATTCAGTCTCCG for U4 pre-snRNA. We used SHAPE MaP buffer (50 mM Tris-HCl pH 8.0, 75 mM KCl, 10 mM DTT, 0.5 mM dNTP, 6 mM MnCl2) and SuperScript II (Invitrogen). Amplicons for snRNAs were generated using gene-specific forward and reverse primers. Importantly, the primers include Nextera adaptors required for downstream library construction. PCR reaction products were cleaned using Monarch PCR&DNA Clean-up Kits. Remaining Illumina adaptor sequences were added using the PCR MasterMix and index primers provided in the NexteraXT DNA Library Preparation Kit (Illumina) according to the manufacturer’s protocol. Libraries were quantified using Qubit (Invitrogen) and BioAnalyzer (Agilent). Amplicons were sequenced on a NextSeq 500/550 platform using a 150 cycle mid-output kit. All sequencing data was analyzed using the ShapeMapper 2 analysis pipeline1. The ‘—amplicon’ and ‘—primers’ flags were used, along with sequences of gene-specific handles PCR primers, to ensure primer binding sites are excluded from reactivity calculations. Default read-depth thresholds of 5000x were used. Analysis of statistically significant reactivity differences between ex vivo and in vivo-determined SHAPE reactivities was performed using the DeltaSHAPE automated analysis tool and default settings2. 1. Busan, S. & Weeks, K.M. Accurate detection of chemical modifications in RNA by mutational profiling (MaP) with ShapeMapper 2. RNA 24, 143-148 (2018). 2. Smola, M.J., Rice, G.M., Busan, S., Siegfried, N.A. & Weeks, K.M. Selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis. Nat Protoc 10, 1643-69 (2015).
2023-09-08 | E-MTAB-13248 | biostudies-arrayexpress
Project description:N6-methylation of 2’-O-methyladenosine (Am) in RNA occurs in eukaryotic cells to generate N6,2’-O-dimethyladenosine (m6Am). Identification of the methyltransferase responsible for m6Am catalysis has accelerated studies on the function of m6Am in RNA processing. While m6Am is generally found in the first transcribed nucleotide of mRNAs, the modification is also found internally within U2 snRNA. However, the writer required for catalyzing internal m6Am formation had remained elusive. By sequencing transcriptome-wide RNA methylation at single-base-resolution, we identified human METTL4 as the writer that directly methylates Am at U2 snRNA position 30 into m6Am. We found that METTL4 localizes to the nucleus and its conserved methyltransferase catalytic site is required for U2 snRNA methylation. By sequencing human cells with overexpressed Mettl4, we determined METTL4’s in vivo target RNA motif specificity. In the absence of Mettl4 in human cells, U2 snRNA lacks m6Am thereby affecting a subset of splicing events that exhibit specific features such as overall 3’ splice-site weakness with certain motif positions more affected than others. This study establishes that METTL4 methylation of U2 snRNA regulates splicing of specific pre-mRNA transcripts.
2020-08-17 | GSE144745 | GEO
Project description:3’ Uridylation Expands miRNA Target Repertoire
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Project description:In this project, we isolate U1 snRNA associated proteins in Arabidopsis thaliana. We used an antisense oligonucleotide specific for the U1 snRNA and analyzed associated proteins by mass spectrometry. As a control, the same experiments were performed with U2 snRNA- and lacZ-specifc antisense oligonucleotides.
2023-12-04 | PXD045484 | Pride
Project description:CG14906 (mettl4) mediates m6A methylation of U2 snRNA in Drosophila
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Project description:We identified U2 as the substrate of mettl4 in fly
2020-04-30 | GSE143958 | GEO
Project description:METTL4 catalyzes m6Am methylation in U2 snRNA to regulate pre-mRNA splicing
| PRJNA604769 | ENA
Project description:The La-related protein LARP7 has been mainly described as a component of the 7SK small nuclear ribonucleoprotein (snRNP) complex, which negatively regulates RNA polymerase II by sequestering the positive transcription elongation factor b (P-TEFb). In our studies, we discovered a novel, 7SK snRNP-independent function of LARP7. We show that LARP7 interacts with the U6 spliceosomal RNA as well as with the small nucleolar RNAs (snoRNAs) directing the 2'-O-methylations of U6. To investigate the relevance of this interaction, U6 or U2 snRNAs were purified from total RNA by pulldown of biotinylated antisense oligonucleotides and the occurence of 2’-O-methylations was investigated by RiboMeth-seq analysis. A comparison between U6 and U2 snRNA isolated from HEK293 wildtype or LARP7 knockout cell lines revealed that 2’-O-methylations of the U6 snRNA are specifically lost in the absence of LARP7. Alazami syndrome is a form of primary dwarfism associated with mutations in the LARP7 gene. RiboMeth-seq analyses performed with RNA isolated from blood samples of two Alazami patients or healthy parents as well as from B-lymphoblastoid cell lines (B-LCLs) derived from an Alazami patient and from a healthy parent confirmed the impact of mutant LARP7 protein variants on the 2’-O-methylation profile of the U6 snRNA.
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