Dataset Information

Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

ABSTRACT: Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We detect positions with increased mutation rates in the reduced and control samples taking the possibility of sequencing/alignment error into account and use replicates to calculate statistical significance based on log likelihood ratio tests. We show that m7G-MaP-seq efficiently detects known m7G modifications in rRNA with mutational rates up to 25% and we map a previously uncharacterised evolutionarily conserved rRNA modification at position 1581 in Arabidopsis thaliana SSU rRNA. Furthermore, we identify m7G modifications in budding yeast, human and arabidopsis tRNAs and demonstrate that m7G modification occurs before tRNA splicing. We do not find any evidence for internal m7G modifications being present in other small RNA, such as miRNA, snoRNA and sRNA, including human Let-7e. Likewise, high sequencing depth m7G-MaP-seq analysis of mRNA from E. coli or yeast cells did not identify any internal m7G modifications.

SUBMITTER: Enroth C

PROVIDER: S-EPMC6847341 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

Enroth Christel C Poulsen Line Dahl LD Iversen Søren S Kirpekar Finn F Albrechtsen Anders A Vinther Jeppe J

Nucleic acids research 20191101 20

Methylation of guanosine on position N7 (m7G) on internal RNA positions has been found in all domains of life and have been implicated in human disease. Here, we present m7G Mutational Profiling sequencing (m7G-MaP-seq), which allows high throughput detection of m7G modifications at nucleotide resolution. In our method, m7G modified positions are converted to abasic sites by reduction with sodium borohydride, directly recorded as cDNA mutations through reverse transcription and sequenced. We det ...[more]

PMID: 31504776

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Project description:Background: RNA modification is one of the epigenetic mechanisms that regulates post-transcriptional gene expression, and abnormal RNA modifications have been reported to play important roles in tumorigenesis. N7-methylguanosine (m7G) is an essential modification at the 5′ cap of human mRNA. However, a systematic and pan-cancer analysis of the clinical relevance of m7G related regulatory genes is still lacking. Methods: We used univariate Cox model and Kaplan-Meier analysis to generate the forest plot of OS, PFI, DSS and identified the correlation between the altered expression of m7G regulators and patient survival in 33 cancer types from the TCGA and GTEx databases. Then, the “estimate” R-package, ssGSEA and CIBERSORT were used to depict the pan-cancer immune landscape. Through Spearman’s correlation test, we analyzed the correlation between m7G regulators and the tumor microenvironment (TME), immune subtype, and drug sensitivity of the tumors, which was further validated in NSCLC. We also assessed the changes in the expression of m7G related regulatory genes in NSCLC with regards to the genetic and transcriptional aspects and evaluated the correlation of METTL1 and WDR4 expression with TMB, MSI and immunotherapy in pan-cancer. Results: High expression of most of the m7G regulators was significantly associated with worse prognosis. Correlation analyses revealed that the expression of majority of the m7G regulators was correlated with tumor immune infiltration and tumor stem cell scores. Drug sensitivity analysis showed that the expression of CYFP1,2 was closely related to drug sensitivity for various anticancer agents (p < 0.001). Analysis of the pan-cancer immune subtype revealed significant differences in the expression of m7G regulators between different immune subtypes (p < 0.001). Additionally, the types and proportions of mutations in METTL1 and WDR4 and their relevance to immunotherapy were further described. Conclusion: Our study is the first to evaluate the correlation between the altered expression of m7G regulators and patient survival, the degree of immune infiltration, TME and drug sensitivity in pan-cancer datasets.

Dataset Information

Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

Publications

Detection of internal N7-methylguanosine (m7G) RNA modifications by mutational profiling sequencing.

Similar Datasets

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