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SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [CRISPR]

ABSTRACT: In this study, we identified SFSWAP as a negative regulator of O-GlcNAc transferase (OGT) intron 4 detention and global pre-mRNA splicing. To do this, we performed CRISPR knockout screens using the Brunello knockout library on Thiamet G (TG)-treated HCT116 cells containing an O-GlcNAc responsive GFP reporter (GFP-β-OGT) integrated at the AAVS1 locus. Knockout of negative regulatory factors which regulate OGT intron 4 detention lead to increased GFP expression. Using replicate screens and further biochemical validation, we identified SFSWAP as one of the top hits affecting OGT intron 4 detention.

ORGANISM(S): Homo sapiens

PROVIDER: GSE277949 | GEO | 2024/10/16

REPOSITORIES: GEO

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SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [RNA-seq I]

Project description:In this study, we identified SFSWAP as a negative regulator of O-GlcNAc transferase (OGT) intron 4 detention and global pre-mRNA splicing using a CRISPR knockout screen in an O-GlcNAc responsive GFP reporter cell line. In order to characterize the functions of the protein, we performed siRNA mediated knockdown of SFSWAP followed by RNA-seq analysis under untreated conditions. Alternate splicing analysis using rMATS (and other tools) revealed enhanced splicing of retained introns and increased inclusion of cassette exons upon knockdown of SFSWAP, supporting an inhibitory role for SFSWAP in splicing.

2024-10-16 | GSE277950 | GEO

SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [RNA-seq II]

Project description:In this study, we identified SFSWAP as a negative regulator of O-GlcNAc transferase (OGT) intron 4 detention and global pre-mRNA splicing using a CRISPR knockout screen in an O-GlcNAc responsive GFP reporter cell line. In order to characterize the functions of the protein, we performed siRNA mediated knockdown of SFSWAP followed by RNA-seq analysis under TG-treated conditions. In addition, we performed the same analysis under UPF1 knockdown conditions to stabilize NMD targeted transcripts. Alternate splicing analysis using rMATS (and other tools) revealed enhanced splicing of retained introns and increased inclusion of cassette exons upon knockdown of SFSWAP, supporting an inhibitory role for SFSWAP in splicing.

2024-10-16 | GSE277951 | GEO

CRISPR screen identifies NUDT21 as a regulator of intron detention of the SAM synthetase MAT2A RNA

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2021-05-04 | GSE172217 | GEO

SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing

Project description:SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing

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SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [CRISPR]

Project description:SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [CRISPR]

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Genome-wide changes in OGT-deficient mouse liver tissue

Project description:Over a billion people suffer from chronic liver diseases worldwide, which often leads to fibrosis and then cirrhosis. Treatments for fibrosis remain experimental, in part because no unifying mechanism has been identified that initiates liver fibrosis. O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a pro-survival role under stress in many tissues. Here we report that OGT protects against hepatocyte necroptosis and initiation of liver fibrosis. Decreased O-GlcNAc levels were seen in patients with alcoholic liver cirrhosis and in mice with ethanol-induced liver injury. Liver-specific O-GlcNAc transferase (OGT) knockout (OGT-LKO) mice progressed to liver fibrosis at 10 weeks of age. OGT-deficient hepatocytes underwent necroptosis. These findings identify OGT as a key suppressor of hepatocyte necroptosis and OGT-LKO mice may serve as an effective spontaneous genetic model of liver fibrosis.

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SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [RNA-seq I]

Project description:SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [RNA-seq I]

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SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [RNA-seq II]

Project description:SFSWAP is a negative regulator of OGT intron detention and global pre-mRNA splicing [RNA-seq II]

| PRJNA1164795 | ENA

Truncation of the TPR domain of OGT alters substrate and glycosite selection

Project description:Nucleocytoplasmic O-linked N-acetylglucosamine (O-GlcNAc) is an essential post-translational modification that is installed to thousands of protein substrates by O-GlcNAc transferase (OGT). Substrate selection by OGT and its isoforms is primarily mediated by the tetratricopeptide repeat (TPR) domain, yet the impact of truncations to the TPR domain on substrate and glycosite selection remains unresolved. Here, we report the effects of TPR truncations on the substrate and glycosite selection of OGT against the model protein GFP-JunB and the surrounding O-GlcNAc proteome in U2OS cells. Truncation of the TPR domain of OGT maintains glycosyltransferase activity but alters subcellular localization in cells. Examination of the glycoproteome across the TPR truncations revealed the broadest substrate activity from the canonical nucleocytoplasmic OGT, with the greatest changes in O-GlcNAc occurring on proteins associated with mRNA splicing processes. Glycosite analysis revealed alterations to the O-GlcNAc consensus sequence globally and differential glycosite selectivity on GFP-JunB as a function of the OGT TPR isoform. This dataset provides a foundation to analyze how perturbations to the TPR domain and expression of OGT isoforms affects the glycosylation of substrates, which will be critical for future protein engineering of OGT, the biology of OGT isoforms, and diseases associated with the TPR domain of OGT.

2021-11-01 | PXD028141 | Pride

TET2 and TET3 regulate GlcNAcylation and H3K4 methylation through OGT and SET1/COMPASS

Project description:TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation. ChIP-Seq experiments were performed on Illumina HiScanSQ sequencer in wild-type HEK293T cells for H3K4me3 histone marks, O-GlcNAc and HCF1, for HT-TET2, HT-TET3 and HT-OGT in HEK293T cells overexpressing those three fusion proteins and in TET2 Kd HEK293T cells for H3K4me3 histone marks. ChIP-Seqs were also performed in mouse bone marrow tissues for H3K4me3 histone marks, O-GlcNAc, endogenous Tet2 and in Tet2 Ko bone marrow tissues for H3K4me3 histone marks.

2013-02-07 | E-GEOD-36620 | biostudies-arrayexpress

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