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Heterogeneous Expression of Alternatively Spliced lncRNA mediates Vascular Smooth Cell Plasticity

ABSTRACT: Heterogeneous Expression of Alternatively Spliced lncRNA mediates Vascular Smooth Cell Plasticity

PROVIDER: PRJNA869091 | ENA |

REPOSITORIES: ENA

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Heterogeneous Expression of Alternatively Spliced lncRNA mediates Vascular Smooth Cell Plasticity

Project description:9p21 locus polymorphisms have the strongest correlation with coronary artery disease, but as a non-coding locus, disease connection is enigmatic. The lncRNA ANRIL found in 9p21 may regulate vascular smooth muscle cell (VSMC) phenotype to contribute to disease risk. We observed significant variability in induced pluripotent stem cell-derived VSMCs from patients homozygous for risk versus isogenic knockout or non-risk haplotypes. Sub-populations of risk haplotype cells exhibited variable morphology, proliferation, contraction, and adhesion. When sorted by adhesion, risk VSMCs parsed into synthetic and contractile sub-populations, i.e., weakly adherent and strongly adherent, respectively. >90% of differentially expressed genes co-regulated by haplotype and adhesion were associated with Rho GTPases, i.e., contractility. Weakly adherent sub-populations expressed more short isoforms of ANRIL, and when overexpressed in knockout cells, ANRIL suppressed adhesion, contractility, and αSMA expression. These data are the first to suggest that variable lncRNA penetrance may drive mixed functional outcomes that confound pathology.

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Mutations primarily alter the inclusion of alternatively spliced exons

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Mutations primarily alter the inclusion of alternatively spliced exons

Project description:Systematic mutagenesis has revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, suggesting that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between sequence variation and exon inclusion across the genome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across the genome but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.

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Gene expression profiling of Capan-1 and Pt45P1 cells treated or not with alternatively spliced tissue factor (asTF)

Project description:The effects of alternatively spliced tissue factor (asTF) were measured by gene expression profiling of Capan-1 and Pt45P1 cells.

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Gene expression profiling of Capan-1 and Pt45P1 cells treated or not with alternatively spliced tissue factor (asTF)

Project description:The effects of alternatively spliced tissue factor (asTF) were measured by gene expression profiling of Capan-1 and Pt45P1 cells. 4 samples: Capan-1 cells +/- asTF, Pt45P1 cells +/- asTF.

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An alternatively spliced variant of METTL3 mediates tumor suppression in hepatocellular carcinoma

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Critical role of the hydrogen peroxide-responsive nuclear kinase CK1-alpha-LS in vascular cell activation

Project description:CK1-alpha-LS was knocked down in human coronary artery smooth muscle cells. Gene level and exon level changes in expression were assessed. CK1-alpha-LS was knocked down in human coronary artery smooth muscle cells using retrovirus containing shRNA targeting the alternatively spliced L-insert.

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Validation of alternatively spliced protein isoforms in human mesenchymal stem cells

Project description:Alternative splicing of mRNA diversifies the function of human proteins, with tissue- and cell-specific protein isoforms being the most difficult to validate. While transcriptomic experiments enable the detection of many alternatively spliced transcripts, it is not known if these transcripts have protein-coding potential. We recently published the PG Nexus pipeline, which facilitates high confidence validation of exons and exon-exon junctions of spliced transcripts by integrating transcriptomics and proteomics data. Using the PG Nexus, we analyzed undifferentiated human mesenchymal stem cells and compared the number of protein isoforms validated using different protein sequence database, including public online databases and RNA-seq derived databases. With significant overlaps with other databases, we identified 8,011 exons and 3,824 splice junctions with the Ensembl database. Both exonic and junction peptides were important for protein isoform validation. The Ensembl database consistently outperformed the other data sources, but predicted open reading frames from RNA-seq derived transcripts were comparable, with only 6 less splice junctions validated. Using proteotypic and isoform-specific peptides, we validated 462 protein isoforms. This number increases to 1,083 if multiple proteotypic peptides per protein are included. Multiplexing proteotypic peptides in SRM assays or similar experiments will increase the confidence and coverage of protein isoform validation experiments.

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