CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE255814 | GEO | 2024/02/15
REPOSITORIES: GEO
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Project description:CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants
| PRJNA1076575 | ENA
Project description:Dilated cardiomyopathy (DCM), defined by left ventricular (LV) enlargement associated with impaired cardiac performance, is a major cause of heart failure (HF). This results in a dilated, thin-walled left ventricle that fails to supply sufficient blood to the body. Truncating variants in TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory DCM. The mechanisms for how TTNtv lead to cardiac dilation are unclear. Here, we show that reduction of Ttn expression by shRNA (Ttn shRNA) generated DCM in both mouse and rat. Ttn shRNA transduced mice developed typical DCM manifestations including impaired cardiac performance, enlarged LV and reduced LV wall thickness. Gene profiling indicates cardiac metabolism, cell proliferation and survival related genes are significantly dysregulated in Ttn shRNA-induced DCM. TUNEL assay showed Ttn shRNA induced a significant increase of cardiac cell apoptosis. A screen of 15 dysregulated downstream genes identified candidates, including Esrra, Esrrb and Yy1 significantly suppressed Ttn shRNA-induced cardiac dilation and/or DCM. Ttn shRNA induced cardiac cell apoptosis was ameliorated by Yy1. Importantly, by inducing D-type cyclin, Yy1 initiated cardiomyocyte cell cycle reentry facilitating the restoration of cardiac performance. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically enhancing cardiac cell proliferation and survival.
2020-03-26 | GSE112319 | GEO
Project description:Prp45 is a budding yeast NineTeen-Complex associated factor, which plays a role in pre-mRNA splicing. prp45(1-169) is a temperature-sensitive truncation allele expressing only the first 169 amino acids (the full-length protein consists of 379 amino acids). RNA-seq analysis was performed to determine changes in transcription and splicing in cells with prp45(1-169) allele prepared by the CRISPR/Cas9 method as a scarless truncation, i.e. with no tags and with preserved endogenous 3' UTR. Total RNA was isolated by combining phenol-chlorophorm extraction with MasterPure Yeast RNA Purification Kit (Epicentre). Ribodepletion, library preparation and sequencing were performed by BGI Genomics.
2024-08-30 | E-MTAB-5334 | biostudies-arrayexpress
Project description:We recently discovered that human neutrophils express immunomodulatory glycoproteins carrying unusual and highly truncated paucimannosidic N-glycans (Man1-3GlcNAc2Fuc0-1), but their biosynthesis remains elusive. Guided by the well-characterised truncation pathway in invertebrates and plants in which the N-acetyl-β-D-hexosaminidase (Hex) isoenzymes catalyse paucimannosidic protein (PMP) formation, we here set out to test if the homologous human Hex α and β subunits encoded by HEXA and HEXB drive a similar truncation pathway in human neutrophils. To this end, we performed quantitative glycomics and glycoproteomics of several CRISPR-Cas9-edited Hex-disrupted neutrophil-like HL-60 mutants (HEXA-/- and HEXB-/-) and matching unedited cell lines. Hex disruption was validated using next-generation sequencing, ELISA, proteomics and Hex activity assays. Excitingly, all Hex-disrupted mutants displayed significantly reduced levels of paucimannosylation, particularly of Man2-3GlcNAc2Fuc1, relative to unedited HL-60 suggesting that both HEXA and HEXB contribute to PMP formation via a hitherto unexplored truncation pathway in neutrophils. Quantitative N-glycomics indeed demonstrated reduced utilisation of a putative non-canonical truncation pathway in favour of the canonical elongation pathway in all Hex-disrupted mutants relative to unedited controls. Quantitative glycoproteomics recapitulated the truncation-to-elongation switch in all Hex-deficient mutants and showed a pronounced switch for N-glycoproteins co-trafficking with Hex to the azurophilic granules of neutrophils such as myeloperoxidase. Finally, we supported the Hex-PMP relationship by documenting that primary neutrophils isolated from an early-onset Sandhoff disease patient (HEXB-/-) displayed dramatically reduced paucimannosylation relative to neutrophils from an age-matched unaffected donor. We conclude that both human Hex α and β mediate PMP formation via a putative non-canonical truncation pathway in neutrophils.
2023-03-11 | PXD027664 | Pride
Project description:Here, we report an O-glycopeptide truncation strategy for the characterization of protein O-GalNAcylation in biological samples. The O-glycopeptide truncation strategy utilizes another protease or O-glycopeptidase for targeted cleavage of tryptic and enriched O-glycopeptides, which increases the analytical coverage of O-GalNAc glycopeptides and glycoproteins. Tryptic O-glycopeptides covered with dense O-glycan clusters and terminal sialic acids could be well isolated by the hydrophilic-based enrichment approaches. And the enriched O-glycopeptides are then enzymatically truncated into shorter or less multiply O-glycosylated peptides, which are more favorable for MS detection and database search in general bottom-up glycoproteomics. We exploit different enzymatic schemes in the O-glycopeptide truncation strategy for large-scale O-glycoproteome analysis. Ultimately, we comprehensively identify nearly 2000 O-glycopeptides from 391 glycoproteins in a total of 75 µL human serum. Moreover, the truncated O-glycopeptides contribute to site-specific O-glycosylation characterization in stepped high-energy collisional dissociation fragmented LC-MS/MS. Together, the O-glycopeptide truncation strategy has great potential to facilitate the in-depth study of O-GalNAc glycoproteomics in biological samples.
2023-05-13 | PXD041041 | JPOST Repository
Project description:Mutations in AT-rich interactive domain-containing protein 1A (ARID1A) cause Coffin-Siris syndrome (CSS), a rare genetic disorder that results in mild to severe intellectual disabilities. However, the biological role of ARID1A in the brain remains unclear. In this study, we report that the haploinsufficiency of ARID1A in excitatory neurons causes cognitive impairment and defects in hippocampal synaptic transmission and dendritic morphology in mice. Similarly, human embryonic stem cell-derived excitatory neurons with deleted ARID1A exhibit fewer dendritic branches and spines, and abnormal electrophysiological activity. Importantly, supplementation of acetate, an epigenetic metabolite, can ameliorate the morphological and electrophysiological deficits observed in mice with Arid1a haploinsufficiency, as well as in ARID1A-null human excitatory neurons. Mechanistically, transcriptomic and ChIP-seq analyses demonstrate that acetate supplementation can increase the levels of H3K27 acetylation at the promoters of key regulatory genes associated with neural development and synaptic transmission. Collectively, these findings support the essential roles of ARID1A in the excitatory neurons and cognition and suggest that acetate supplementation could be a potential therapeutic intervention for CSS.
| S-SCDT-10_15252-EMMM_202215795 | biostudies-other
Project description:miRNA levels depend on both biogenesis and turnover. The methyltransferase HEN1 stabilizes plant miRNAs, animal piRNAs, and siRNAs in both kingdoms via 3' terminal methylation. Loss of HEN1 in plants results in non-templated oligo-uridylation and accelerated degradation of miRNAs. In hen1 mutants from Arabidopsis and rice, we found that the patterns of miRNA truncation and uridylation differ substantially among miRNA families, but such patterns for the same miRNA are conserved between species. miR166 and miR163 are truncated predominantly to ~17 and ~16 nt, and subsequently recover via uridylation to approximately their original sizes, 21 and 24 nt, suggesting that in these cases miRNA truncation triggers uridylation. miR171 is untruncated but uridylated to 22 nt in hen1 mutants, gaining the ability to trigger production of phased, secondary siRNAs. Truncated and tailed variants were bound by ARGONAUTE1 (AGO1) in hen1, implying that these events occur while miRNAs are still bound by AGO1. Unexpectedly, a portion of miR158 in wildtype remains unmethylated and thus subject to uridylation and destabilization, suggesting that plants naturally utilize miRNA methylation to modulate miRNA accumulation. Our results suggest that the AGO1-containing RISC complex may undergo programming to reflect each bound miRNA, determining a defined, distinct decay destiny.
2013-07-22 | GSE35562 | GEO
Project description:Mutations in AT-rich interactive domain-containing protein 1A (ARID1A) cause Coffin-Siris syndrome (CSS), a rare genetic disorder that results in mild to severe intellectual disabilities. However, the biological role of ARID1A in the brain remains unclear. In this study, we report that the haploinsufficiency of ARID1A in excitatory neurons causes cognitive impairment and defects in hippocampal synaptic transmission and dendritic morphology in mice. Similarly, human embryonic stem cell-derived excitatory neurons with deleted ARID1A exhibit fewer dendritic branches and spines, and abnormal electrophysiological activity. Importantly, supplementation of acetate, an epigenetic metabolite, can ameliorate the morphological and electrophysiological deficits observed in mice with Arid1a haploinsufficiency, as well as in ARID1A-null human excitatory neurons. Mechanistically, transcriptomic and ChIP-seq analyses demonstrate that acetate supplementation can increase the levels of H3K27 acetylation at the promoters of key regulatory genes associated with neural development and synaptic transmission. Collectively, these findings support the essential roles of ARID1A in the excitatory neurons and cognition and suggest that acetate supplementation could be a potential therapeutic intervention for CSS.
| S-EPMC9728054 | biostudies-literature
Project description:FGFR2 C-terminal truncation in cancer
| PRJEB42514 | ENA
Project description:Mutations in the sarcomeric protein titin are a major cause of human dilated cardiomyopathy. We have developed a knock-in mouse model that imitated a previously identified titin truncation mutation. The heterotygous Ttn-deficient mice develop features of DCM and therefore recapitulate the human phenotype. To investigate the role of microRNAs in titin-based heart failure, we performed a miRNA screen in heterozygous Ttn-deficient mice and their wildtype littermate controls.
2017-01-31 | GSE90972 | GEO