Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1145569 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR30167907_1.fastq.gz | Fastqsanger.gz | |||
| SRR30167907_2.fastq.gz | Fastqsanger.gz | |||
| SRR30167908_1.fastq.gz | Fastqsanger.gz | |||
| SRR30167908_2.fastq.gz | Fastqsanger.gz | |||
| SRR30167909_1.fastq.gz | Fastqsanger.gz |
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Project description:Pyruvate kinase M isoform 2 (PKM2) is preferentially expressed in nearly all cancers. It primarily functions as the last enzyme in glycolysis, but has other reported non-canonical functions, including recruiting transcription factors to oncogenes, and phosphorylating proteins. We previously described antisense oligonucleotides that disrupt alternative splicing of PKM pre-mRNA (PKM-ASOs), resulting in PKM2-to-PKM1 isoform switching in hepatocellular carcinoma (HCC), which reduces HCC growth. PKM1 has higher enzymatic activity than PKM2, which potentially drives metabolism away from macromolecule synthesis, and may explain decreased HCC growth upon PKM-ASO treatment. As PKM-ASOs also reduce PKM2 levels, how PKM splice-switching inhibits HCC cell proliferation was unclear. Here, we characterized the individual consequences of altering PKM1 or PKM2 protein levels in HCC, and observed that reducing PKM2 alone was sufficient to decrease HCC cell proliferation, whereas overexpressing PKM1 had no effect. Moreover, increasing PK activity via a small-molecule PKM2 activator had no effect on HCC cell proliferation, suggesting that PKM-ASOs affect PKM2’s non-metabolic functions. Transcriptomic and RT-qPCR analyses of HCC cells treated with PKM-ASO or PKM2-siRNA revealed upregulation of dual-specificity phosphatase 2 (DUSP2) and other related DUSPs, which act directly on ERK1/2 in the MAPK signaling pathway. Luciferase reporter assays demonstrated that PKM-ASO treatment activated the DUSP2 promoter, which correlated with decreased ERK1/2 phosphorylation. Lenvatinib is a second-line HCC therapy that indirectly reduces ERK1/2 phosphorylation, and combined treatment with PKM-ASOs inhibited proliferation of HCC cells more than either treatment alone. In summary, our results reveal a mechanism by which PKM-ASOs affect PKM2 dependency in HCC.
2025-08-09 | GSE274520 | GEO
Project description:Mitotic exit requires extensive dephosphorylation of Ser/Thr residues by the PP1 and PP2A-B55 protein phosphatases in human cells. Several aspects of this are poorly understood including specific substrates and determinants of phosphatase specificity. Here we develop a novel in vitro assay, MRBLE-dephos, that allows multiplexing of dephosphorylation reactions to determine phosphatase specificity. Using MRBLE-dephos, we establish amino acid preferences of the residues surrounding the phosphorylation site for PP1 and PP2A-B55, which reveals common and unique preferences for the two phosphatases. We use specific inhibition of PP1 and PP2A-B55 in mitotic exit lysates coupled with quantitative phosphoproteomics to identify more than 2000 regulated phosphorylation sites and integrate this with mitotic interactomes to obtain a comprehensive map of mitotic dephosphorylation events. Importantly, the sites dephosphorylated during mitotic exit reveal key signatures that are consistent with the MRBLE-dephos results. We use these insights to specifically alter INCENP dephosphorylation kinetics at mitotic exit resulting in defective cytokinesis underscoring the biological relevance of our determined specificity principles. Finally, we provide a comprehensive characterization of PP1 binding motifs and show how these can shape dephosphorylation and how PP1 primes its own association with these motifs at mitotic exit. Collectively we provide a framework for understanding mitotic exit regulation by dephosphorylation and novel approaches to dissect protein phosphatase specificity.
2023-05-12 | MSV000091928 | MassIVE
Project description:Circular RNAs (circRNAs) constitute an abundant class of covalently closed non-coding RNA molecules that are formed by backsplicing from eukaryotic protein-coding genes. Recent studies have shown that circRNAs can act as microRNA or protein decoys as well as transcriptional regulators. However, the functions of most circRNAs are still poorly understood. Because circRNA sequences overlap with their linear parent transcripts, depleting specific circRNAs without affecting host gene expression remains a challenge. Here, we assessed the utility of LNA-modified antisense oligonucleotides (ASOs) to knock down circRNAs for loss-of-function studies. We identified 5807 circRNAs in total RNA sequencing data from 4 liver cancer cell lines and used the back splice junction (BSJ) sequences of 7 validated circRNAs as target sites for designing different LNA-modified ASOs for circRNA knockdown. We found that while most RNase H-dependent gapmer ASOs mediate effective knockdown of their target circRNAs, some gapmers reduce the levels of the linear parent transcript and may also cause degradation of unintended off-targets. The circRNA targeting specificity can be enhanced using design-optimized gapmer ASOs or LNA/DNA mixmer ASOs, which display potent and specific circRNA knockdown with a minimal effect on the host genes or predicted off-targets. In summary, our results demonstrate that LNA-modified ASOs complementary to BSJ sequences mediate robust knockdown of circRNAs in vitro and, thus, represent a useful tool to explore the biological roles of circRNAs in loss-of-function studies in cultured cells and animal models.
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Project description:FDA-approved BRAF inhibitors produce high response rates and improve overall survival in patients with BRAF V600E/K mutant melanoma, but are linked to pathologies associated with paradoxical ERK1/2 activation in wild-type BRAF cells. To overcome this limitation, a next-generation paradox breaking RAF inhibitor (PLX8394) has been designed. Here we show that by using a quantitative reporter assay, PLX8394 rapidly suppressed ERK1/2 reporter activity and growth of mutant BRAF melanoma xenografts. Ex vivo treatment of xenografts and use of a patient-derived explant system (PDeX) revealed that PLX8394 suppressed ERK1/2 signaling and elicited apoptosis more effectively than the FDA-approved BRAF inhibitor, vemurafenib. Furthermore, PLX8394 was efficacious against vemurafenib-resistant BRAF splice-variant expressing tumors and reduced splice-variant homodimerization. Importantly, PLX8394 did not induce paradoxical activation of ERK1/2 in wild-type BRAF cell lines or PDeX. Continued in vivo dosing of xenografts with PLX8394 led to the development of acquired resistance via ERK1/2 reactivation through heterogeneous mechanisms; however, resistant cells were found to have differential sensitivity to ERK1/2 inhibitor. These findings highlight the efficacy of a paradox-breaking selective BRAF inhibitor and the use of PDeX system to test efficacy of therapeutic agents.
2022-01-24 | GSE180567 | GEO
Project description:TOX4 is one of the regulatory proteins of PP1 phosphatases with poorly understood functions. Here we show that chromatin occupancy pattern of TOX4 resembles that of RNA polymerase II (Pol II), and its loss increases cellular level of C-terminal domain (CTD) phosphorylated Pol II but mainly decreases Pol II occupancy on promoters. In addition, elongation rate analyses by 4sUDRB-seq suggest that TOX4 restricts pause release and early elongation but promotes late elongation. Moreover, TT-seq analyses indicate that TOX4 loss mainly decreases transcriptional output. Mechanistically, TOX4 may restrict pause release through facilitating CTD serine 2 and DSIF dephosphorylation, and promote Pol II recycling and reinitiation through facilitating CTD serines 2 and 5 dephosphorylation. Furthermore, among the PP1 phosphatases, TOX4 preferentially binds PP1α and is capable of facilitating Pol II CTD dephosphorylation in vitro. These results lay the foundation for a better understanding of the role of TOX4 in transcriptional regulation.
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Project description:Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well-established that single-stranded, phosphorothioate modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus rendering the majority of ASO as inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ~2-fold. Bulk ASO uptake is 5-fold higher in GOLGA8 overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.
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Project description:Light-induced phosphorylation is necessary and essential for the degradation of phytochrome-interacting factors (PIFs), the central repressors of photomorphogenesis. Although the kinases responsible for PIF phosphorylation have been extensively studied, the phosphatases underlying PIF dephosphorylation are largely unknown. Here, we real that mutation of FyPP1 and FyPP3, two catalytic subunits of PP6 phosphatases, promoted photomorphogenesis of seedlings in the dark. PP6 and PIFs functioned synergistically to repress photomorphogenesis. FyPP1 and FyPP3 directly interacted with and dephosphorylated PIF3 and PIF4. The light-induced degradation of PIF4 and the PIF transcriptional activities were dependent on PP6 activity. These data demonstrate that PP6 phosphatases repress photomorphogenesis through regulation of PIF phosphorylation, protein stability and transcriptional activity.
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