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A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing [RNA-Seq]

ABSTRACT: miRNAs are regulatory transcripts established as repressors of mRNA stability and translation. Here we demonstrate that an oncomiR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of direct binding between miR-10b and U6, in situ visualizations of miR-10b and U6 co-localization in glioma cells and tumor tissues, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. The effects on U6 result in splicing alterations, illustrated by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a cancer-associated miRNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE182280 | GEO | 2022/01/19

REPOSITORIES: GEO

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A nuclear function for an oncogenic microRNA as a modulator of snRNA and splicing [CLEAR-CLIP]

Project description:miRNAs are regulatory transcripts established as repressors of mRNA stability and translation. Here we demonstrate that an oncomiR-10b binds to U6 snRNA, a core component of the spliceosomal machinery. We provide evidence of direct binding between miR-10b and U6, in situ visualizations of miR-10b and U6 co-localization in glioma cells and tumor tissues, and biochemical co-isolation of miR-10b with the components of the spliceosome. We further demonstrate that miR-10b modulates U6 N-6-adenosine methylation and pseudouridylation, U6 binding to splicing factors SART3 and PRPF8, and regulates U6 stability, conformation, and levels. The effects on U6 result in splicing alterations, illustrated by the altered ratio of the isoforms of a small GTPase CDC42, reduced overall CDC42 levels, and downstream CDC42 -mediated effects on cell viability. We, therefore, present an unexpected intersection of the miRNA and splicing machineries and a new nuclear function for a cancer-associated miRNA.

2022-01-19 | GSE182281 | GEO

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

Project description:The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926A>C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5’-splice site (5’SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5’SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

2024-03-01 | PXD043645 | Pride

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5'-splice-site selection causing tissue-specific defects

2024-02-16 | GSE236702 | GEO

PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects

Project description:Mutations in the carboxy terminal of the core spliceosome factor PRPF8 cause autosomal dominant retinitis pigmentosa (RP) 13. Comprehensive cellular, biochemical, and molecular investigations of iPSC-derived retinal organoids, retinal pigment epithelium (RPE) and kidney organoids from four patients carrying the pathogenic PRPF8 RP type 13 c.6926A>C (p.H2309P) heterozygous missense mutation, revealed retinal tissue-specific effects including lower splicing specificity, ciliary abnormalities, altered apical-basal polarity, rod degeneration and loss of photoreceptors. The p.H2309P mutation affected the 5’ splice site recognition by PRPF8 of transcripts encoding ciliary proteins, altered spliceosome kinetics and organisation of nuclear speckles as well as PRPF8 binding to spliceosomal U6 snRNAs and snoRNAs, leading to accumulation of unspliced poly A+ mRNAs specifically in RPE cells and retinal organoids. Together these data provide the most comprehensive characterisation of splicing factor causing RP disease, providing molecular insights into the tissue specificity of pathomechanisms and informing future therapeutic approaches.

2024-02-29 | GSE235866 | GEO

Identification of SART3 as a pre-miR-34a–binding protein

Project description:MicroRNAs (miRNAs or miRs) are small, noncoding RNAs that are implicated in the regulation of nearly all biological processes. Global miRNA biogenesis is altered in many cancers and RNA-binding proteins (RBPs) have been shown to play a role in this process, presenting a promising avenue for targeting miRNA dysregulation in disease. miR-34a exhibits tumor-suppressive functions by targeting cell cycle regulators CDK4/6 and anti-apoptotic factor Bcl-2, among other regulatory pathways such as Wnt, TGF-, and Notch signaling. Many cancers show downregulation or loss of miR-34a, and synthetic miR-34a supplementation has been shown to inhibit tumor growth in vivo; however, the post-transcriptional mechanisms by which miR-34a is lost in cancer are not entirely understood. Here, we have used a proteomics-mediated approach to identify Squamous cell carcinoma antigen recognized by T-cells 3 (SART3) as a putative pre-miR-34a-binding protein. SART3 is a spliceosome recycling factor and nuclear RBP with no previously reported role in miRNA regulation. We demonstrate that SART3 binds pre-miR-34a with specificity over pre-let-7d and begin to elucidate a new functional role for this protein in non-small lung cancer cells. Overexpression of SART3 led to increased miR-34a levels, downregulation of the miR-34a target genes CDK4 and CDK6, and cell cycle arrest in the G1 phase. In vitro binding studies showed that the RNA-recognition motifs within the SART3 sequence are responsible for selective pre-miR-34a binding. Collectively, our results present evidence for an influential role of SART3 in miR-34a biogenesis and cell cycle progression.

2019-10-14 | PXD015278 | Pride

SILAC APMS anlysis of TSSC4 interactome

Project description:U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo an intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before a next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a new component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.

2021-06-11 | PXD024929 | Pride

Stress-induced lncRNA LAS3 fosters fitness of cancer cells by regulating SART3-mediated spliceosome recycling

Project description:Long non-coding RNAs (lncRNAs) represent a novel class of anti-cancer therapeutic targets. Hypoxia-induced lncRNAs are associated with the aggressive tumor phenotypes and might serve as putative drug targets. Here, we unraveled lncRNAs whose expression is upregulated in hypoxic breast tumors. One of the hypoxia-induced lncRNA, LAS3 (LncRNA Associated to SART3), is commonly upregulated not only in all breast cancer subtypes, but also in several types of epithelial cancers. LAS3 expression is driven by the stress-induced JNK/c-JUN pathway, which is frequently activated in human cancer. By pull down of LAS3 coupled to mass spectrometry-based proteomics, we identified SART3, a component of the splicing machinery, as a LAS3-interacting partner. In a second proteomics experiment, pull down of SART3-containing complexes from MCF10A cells treated with either scramble, or LAS3-specific GapmeRs showed that LAS3 regulates splicing efficiency by triggering SART3 dissociation from the U4/U6 snRNP during the recycling phase of the spliceosome cycle. Finally, differential shotgun analysis of MDA-MB-231/tet-shLAS3 cells allowed us to quantify expression of 2,940 proteins. Here, genes with significant intron retention showed decreased protein expression levels, indicating that widespread LAS3-mediated intron retention disrupts open reading frame integrity leading to stochastic decrease of protein expression and decreased fitness of cancer cells. Together, our data show that LAS3 is essential for growth of LAS3-positive triple negative breast tumors and indicate that LAS3 inhibition might be a suitable therapeutic approach for breast cancer treatment.

2020-05-26 | PXD013334 | Pride

Basal-A Triple Negative Breast Cancer Cells Selectively Rely on RNA Splicing for Survival

Project description:Development of targeted therapies for triple-negative breast cancer (TNBC) has failed at least in part because of the heterogeneity of these poor prognosis cancers. To identify common dependencies of basal-like TNBCs, we performed a targeted siRNA lethality screen in 7 human breast cancer cell lines focusing on 154 previously identified dependency genes of one basal-A TNBC line. Thirty genes were shared basal-A TNBC dependencies. Genes for proteins involved in RNA splicing, in particular those associated with the U4/U6.U5 tri-snRNP complex (e.g. PRPF8, PRPF38A), were prominent shared dependencies. Spliceosome genes were commonly up-regulated in primary basal-like TNBCs. Knockdown of PRPF8 or PRPF38A led to widespread intronic retention, expression of immune system genes and aberrant splicing of transcripts involved in protein translation, proteasome function, mitosis and apoptosis, including the TNBC dependency gene MCL1. These transcripts were susceptible to inhibition of RNA splicing more generally, since they were similarly affected by the RNA splicing inhibitor drug E7107. In particular, E7107 caused splicing of MCL1 to its pro-apoptotic splicing variant in multiple cell lines, killed MCL1-dependent basal-A cells in vitro, and suppressed the growth of 2 basal-A cell-lines and 1 of 2 patient-derived basal-like TNBC xenografts in vivo. Thus, TNBCs critically rely on RNA splicing and could be susceptible to RNA splicing inhibitors.

2018-01-01 | GSE90519 | GEO

SART3 is a Novel Rme2s Reader

Project description:Arginine methylation is a ubiquitous post-translational modification involved in many biological processes such as transcription, cell signaling and RNA splicing. Tudor domains by far are the major effector domain family for arginine methylation mark recognition. Here we characterize SART3 as a novel selective reader for symmetric dimethylarginine (Rme2s) marks. SART3 harbors a series of HAT (Half-a-TPR) repeats that are aromatic-rich, and it is this region that binds Rme2s motifs. An analysis of the reported structure of the HAT repeats of SART3 identified a putative aromatic cage that potentially “read” the Rme2s-modified motifs, and the key aromatic residues required for Rme2s recognition were identified. We further confirm that this Rme2s reader ability is important for SART3-mediated RNA splicing. Together, our studies revealed that the evolutionarily conserved SART3 HAT domain was a novel reader of Rme2s mark involved in RNA splicing.

2024-10-01 | GSE246489 | GEO

Gene expression profile upon miR-10b overexpression or knockdown

Project description:miR-10b precusor or miR-10b inhibitor was used to overexpress or knockdown miR-10b expression in PANC-1 cells. Microarray analysis was used to characterize the changes in gene expression profiles of PANC-1 upon miR-10b overexpression or knockdown

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