Gene Expression
Nebulas
Gene Expression NebulasSummary: Nopp140-chaperoned 2’-O-methylation of small nuclear RNAs in Cajal bodies ensures splicing fidelity
Overall Design: Spliceosomal small nuclear RNAs (snRNAs) are modified by small Cajal body (CB) specific ribonucleoproteins (scaRNPs) to ensure snRNP biogenesis and pre-mRNA splicing. However, the function and subcellular site of snRNA modification are largely unknown. We show that CB localization of the protein Nopp140 is essential for concentration of scaRNPs in that nuclear condensate; and that phosphorylation by casein kinase 2 (CK2) at some 80 serines targets Nopp140 to CBs. Transiting through CBs, snRNAs are apparently modified by scaRNPs. Indeed, Nopp140 knockdown-mediated release of scaRNPs from CBs severely compromises 2’-O-methylation of spliceosomal snRNAs, identifying CBs as the site of scaRNP catalysis. Additionally, alternative splicing patterns change indicating that these modifications in U1, U2, U5, and U12 snRNAs safeguard splicing fidelity. Given the importance of CK2 in this pathway, compromised splicing could underlie the mode of action of small molecule CK2 inhibitors currently considered for therapy in cholangiocarcinoma, hematological malignancies, and COVID-19.
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| Extract Protocol: | RNA from the different cell lines was extracted using 500 µl TRIzol Reagent (Ambion) directly on 10-cm dishes (cell confluency ∼80%, ∼1,000,000 cells). Lysed cells in TRIzol were scraped into tubes, extracted twice with chloroform, the RNA was precipitated with 0.7 volume isopropanol after addition of 20 µg glycogen, and resuspended in UltraPure distilled water. RNA concentration and quality were determined by Nanodrop (ratio 260/230 and 260/280 above 1.8) and Agilent 2100 BioAnalyzer (RIN above 8). Total RNA was used for northern blot analysis, RiboMethSeq, and RT-PCR and RNase H analysis. |
| Library Construction Protocol: | For deep sequencing, total RNA was prepared from 3 separate dishes for each sample and shipped to Novogene Corporation Inc. (Sacramento, CA) for cDNA library preparation (250~300 bp inserts) and Illumina sequencing (PE150). The RNA was prepared and sequenced one year apart in the USA and in China in two batches, one for the P1, KD1a, and KD1b cells and one for the P2 and KD2 cells. |
| Molecule Type: | total RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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