Bisulfite Incorporation Hindered Ligation-based Method for Locus-Specific Detection and Quantification of Pseudouridine
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE269170 | GEO | 2024/11/01
REPOSITORIES: GEO
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Project description:Colorectal cancer (CRC) with high rate of mortality is one of the most commonly diagnosed cancers in worldwide. Advanced colorectal cancer is often accompanied by malignant proliferation of tumor cells. Further researches on colorectal cancer proliferation to find new targets and develop new therapeutic regimen are an important direction for colorectal cancer treatment. In this study, genome-wide RNAi screening was used to discover the essential genes associated with colorectal cancer cell proliferation. We found DKC1 (dyskerin pseudouridine synthase 1) promoted CRC proliferation through binding and modifying ribosomal proteins (RPL10A, RPL22L1, RPL34, RPS3) mRNA to increase the mRNAs stability and eventually increasing their expression. Besides, DKC1 is highly expressed in colorectal cancer and its aberrantly high expression is associated with poor prognosis of colorectal cancer patients. These results suggest that DKC1 could be a new therapeutic target for colorectal cancer.
2021-02-08 | PXD018103 | Pride
Project description:Colorectal cancer (CRC) with high rate of mortality is one of the most commonly diagnosed cancers in worldwide. Advanced colorectal cancer is often accompanied by malignant proliferation of tumor cells. Further researches on colorectal cancer proliferation to find new targets and develop new therapeutic regimen are an important direction for colorectal cancer treatment. In this study, genome-wide RNAi screening was used to discover the essential genes associated with colorectal cancer cell proliferation. We found DKC1 (dyskerin pseudouridine synthase 1) promoted CRC proliferation through binding and modifying ribosomal proteins (RPL10A, RPL22L1, RPL34, RPS3) mRNA to increase the mRNAs stability and eventually increasing their expression. Besides, DKC1 is highly expressed in colorectal cancer and its aberrantly high expression is associated with poor prognosis of colorectal cancer patients. These results suggest that DKC1 could be a new therapeutic target for colorectal cancer.
2021-04-30 | GSE147348 | GEO
Project description:Pseudouridine is the most abundant modification occurring on RNA, yet with the exception of a few well-studied RNA molecules little is known about the modified positions and their function(s). Here, we develop M-NM-(-seq, a method for transcriptome-wide quantitative mapping of pseudouridine. We validate M-NM-(-seq with synthetic spike-ins and de novo identification of the vast majority of previously reported pseudouridylated positions. M-NM-(-seq permits discovery of hundreds of novel pseudouridine modifications in human and yeast mRNAs and snoRNAs. Knockdown and knockout of pseudouridine synthases uncovers the cognate PUSs mediating pseudouridine catalysis at these individual novel sites and their target sequence features. In both human and yeast pseudouridine formation on mRNA depends on both site-specific PUSs M-bM-^@M-^S often guided by a specific sequence motif - and snoRNA-guided PUSs. Importantly, upon heat shock in yeast, Pus7-mediated pseudouridylation is induced at >200 sites in diverse mRNAs. Pus7 deletion in yeast leads to decreased recovery from heat shock and decreased RNA levels at otherwise pseudouridylated messages, suggesting a role for pseudouridine in enhancing transcript stability. Pseudouridine stoichiometries in rRNA are highly conserved from yeast to mammals, but are reduced in cells derived from dyskeratosis congenita patients, where the pseudouridine synthase DKC1 is mutated, compared to age matched controls. Our results establish pseudouridine as a ubiquitous and dynamic modification in mRNA, and provide a sensitive, quantitative and transcriptome-wide methodology to address its underlying mechanisms and function. Examination of m6A methylation in human Hek293 and A549 cell lines, in human embryonic stem cells (ESCs) undergoing differentiation to neural progenitor cells (NPCs), in OKMS inducible fibroblasts reprogrammed into iPSC, and upon knockdown of factors using siRNAs or shRNAs.
2014-09-11 | E-GEOD-60047 | biostudies-arrayexpress
Project description:Purpose:Pseudouridine (Ψ) is the most abundant RNA epigenetic modification and plays vital roles in various biological processes. However, its biogenesis and functions in mRNAs remain elusive. Method:Here, we developed an approach for enriching Ψ sites with deep sequencing (edu-Ψ-seq), which utilizes both exoribonuclease and restriction endonuclease activity to eliminate other nucleosides or reads without Ψ modifications. Results:Using edu-Ψ-seq approach in the dyskerin pseudouridine synthase 1 (DKC1) knockdown cells, we identified that more than 100 pseudouridines could be catalyzed by DKC1 in human mRNAs and non-coding RNAs (ncRNAs). Surprisingly, we discovered, for the first time, 10 pseudouridines in mRNAs might be guided by 11 small nucleolar ncRNAs (snoRNAs). Interestingly, we found that SNORA10 could guide DKC1 protein to induce the pseudouridylation of RPL15 mRNA. Importantly, loss of SNORA10 impaired the protein synthesis of RPL15 and inhibited cell proliferation. Conclusion:Overall, our study not only developed a powerful method for detecting Ψ sites but also uncovered another layer of gene expression regulation that involves crosstalk among snoRNAs, mRNA pseudouridylation and protein synthesis.
2024-10-18 | GSE192381 | GEO
Project description:Purpose:Pseudouridine (Ψ) is the most abundant RNA epigenetic modification and plays vital roles in various biological processes. However, its biogenesis and functions in mRNAs remain elusive. Method:Here, we developed an approach for enriching Ψ sites with deep sequencing (edu-Ψ-seq), which utilizes both exoribonuclease and restriction endonuclease activity to eliminate other nucleosides or reads without Ψ modifications. Results:Using edu-Ψ-seq approach in the dyskerin pseudouridine synthase 1 (DKC1) knockdown cells, we identified that more than 100 pseudouridines could be catalyzed by DKC1 in human mRNAs and non-coding RNAs (ncRNAs). Surprisingly, we discovered, for the first time, 10 pseudouridines in mRNAs might be guided by 11 small nucleolar ncRNAs (snoRNAs). Interestingly, we found that SNORA10 could guide DKC1 protein to induce the pseudouridylation of RPL15 mRNA. Importantly, loss of SNORA10 impaired the protein synthesis of RPL15 and inhibited cell proliferation. Conclusion:Overall, our study not only developed a powerful method for detecting Ψ sites but also uncovered another layer of gene expression regulation that involves crosstalk among snoRNAs, mRNA pseudouridylation and protein synthesis.
2024-10-18 | GSE190820 | GEO
Project description:Pseudouridine (Ψ) is the most abundant post-transcriptional RNA modification. Various methods have been developed to achieve locus-specific Ψ detection; however, the existing methods often involve radiolabeling of RNA, require advanced experimental skills and can be time-consuming. Herein we report a radiolabeling-free，qPCR-based method to detect locus-specific Ψs in rRNA and mRNA. This method is based on Ψ chemical adduct (Ψ-CMC) induced mutation/deletion during reverse transcription (RT), leading to qPCR products of different melting temperatures. Utilizing high-throughput sequencing, we demonstrate that such misincorporation is a general feature of Ψ-CMC adduct during our improved RT conditions. We validated this method on known Ψ sites in rRNA and showed that the melting curves correlate with the modification level. Moreover, we successfully detected Ψs in mRNA and lncRNA of different abundance, and identified Ψ synthase that targets mRNA. Our facile method takes only 1.5 days to complete, and with slight adjustment it can be applied to detect other epitranscriptomic marks in the transcriptome.
2017-10-03 | GSE102476 | GEO
Project description:Pseudouridine is an isomer of uridine and is the most common RNA modification in both procaryotes and eucaryotes. It is found in ribosomal, transfer, and other structural RNA as well as in some mRNA and non-coding RNA. We have found abundant pseudouridine in small RNA and their precursors in Arabidopsis.
2023-08-07 | GSE222751 | GEO
Project description:Pseudouridine is an isomer of uridine and is the most common RNA modification in both procaryotes and eucaryotes. It is found in ribosomal, transfer, and other structural RNA as well as in some mRNA and non-coding RNA. We have found abundant pseudouridine in small RNA and their precursors in Arabidopsis.
2023-08-07 | GSE230228 | GEO
Project description:In an effort to produce a mouse model of Mitochondrial Myopathy with Lactic acidosis and Sideroblastic Anemia (MLASA), we knocked out the gene for Pseudouridine synthase 1 (PUS1), an enzyme that modifies uridine to pseudouridine in many cytoplasmic and mitochondrial tRNAs, as well as other cellular RNAs. The Pus1-/- mice are viable, are born at the expected Mendelian frequency, and are non-dysmorphic. The PUS1 mRNA and certain pseudouridine modifications are absent in cytoplasmic and mitochondrial tRNAs in the Pus1-/- mice. The Pus1-/- mice display reduce exercise capacity at 14 weeks, with alterations in muscle morphology, histology, and physiology. Red gastrocnemius muscle from Pus1-/- mice shows reduced number and size of mitochondria and reduced Cytochrome C oxidase activity. Two-condition, two-color experiment: Mouse wild type PUS1 and homozygous mutant PUS1 kidney tissue samples: 4 biological replicates each.
2016-05-16 | E-GEOD-77819 | biostudies-arrayexpress
Project description:In an effort to produce a mouse model of Mitochondrial Myopathy with Lactic acidosis and Sideroblastic Anemia (MLASA), we knocked out the gene for Pseudouridine synthase 1 (PUS1), an enzyme that modifies uridine to pseudouridine in many cytoplasmic and mitochondrial tRNAs, as well as other cellular RNAs. The Pus1-/- mice are viable, are born at the expected Mendelian frequency, and are non-dysmorphic. The PUS1 mRNA and certain pseudouridine modifications are absent in cytoplasmic and mitochondrial tRNAs in the Pus1-/- mice. The Pus1-/- mice display reduce exercise capacity at 14 weeks, with alterations in muscle morphology, histology, and physiology. Red gastrocnemius muscle from Pus1-/- mice shows reduced number and size of mitochondria and reduced Cytochrome C oxidase activity. Two-condition, two-color experiment: Mouse wild type PUS1 and homozygous mutant PUS1 liver tissue samples: 4 biological replicates each.
2016-05-16 | E-GEOD-77820 | biostudies-arrayexpress