Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Identification of direct targets and modified bases of RNA cytosine methyltransferases

ABSTRACT: We report development of a mechanism-based technique, Aza-IP, that utilizes the stable covalent linkage formed in vivo between an RNA methyltransferase (m5C-RMT) and 5-azacytidine (5-aza-C) incorporated within the target RNA to enable specific target enrichment, coupled with high-throughput sequencing to provide target identification. We apply Aza-IP to two enzyme types, DNMT2 and NSUN2, the latter with important roles in fertility, intellectual ability, stem cells and cancer. For both, Aza-IP provided >200-fold enrichment of their known human tRNA targets. For NSUN2, we reveal many novel tRNA and non-coding RNA targets, all linked to NSUN2 nucleolar localization, greatly increasing the potential repertoire underlying loss-of-function pathologies. Strikingly, we observe a C>G transversion ‘signature’ specifically at the target cytosine, which enables the identification of the exact target cytosine within the RNA in the same experiment. The general design of Aza-IP involves nine steps: 1) Expression of an epitope-tagged m5C-RMT derivative (or use of an antibody capable of immuno-precipitating the RNA-bound enzyme), 2) cell growth in the presence of 5-aza-C, which incorporates at low/moderate levels into nascent RNA, 3) cell lysis, 4) immuno-precipitation of the subject m5C-RMT, a portion of which is covalently attached to target RNAs bearing 5-aza-C, 5) stringent washing to remove RNA contaminants, 6) RNA fragmentation, release and purification, 7) ligation of adaptor oligos to the RNA, and the creation of a cDNA library in a manner that enables strand-specific assignments, 8) cDNA sequencing, and 9), mapping and examination of sequence reads to define RNA targets and site of cross-linking/catalysis.

ORGANISM(S): Homo sapiens

SUBMITTER: Vahid Khoddami

PROVIDER: E-GEOD-38957 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Identification of direct targets and modified bases of RNA cytosine methyltransferases.

Khoddami Vahid V Cairns Bradley R BR

Nature biotechnology 20130421 5

The extent and biological impact of RNA cytosine methylation are poorly understood, in part owing to limitations of current techniques for determining the targets of RNA methyltransferases. Here we describe 5-azacytidine-mediated RNA immunoprecipitation (Aza-IP), a technique that exploits the covalent bond formed between an RNA methyltransferase and the cytidine analog 5-azacytidine to recover RNA targets by immunoprecipitation. Targets are subsequently identified by high-throughput sequencing. ...[more]

PMID: 23604283

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Project description:Background: As a widespread post-transcriptional RNA modification, N5-methylcytosine (m5C) is implicated in a variety of cellular responses and processes that regulate RNA metabolism. Despite this, a clear understanding of m5C modification’s role and mechanism in angiogenesis is still lacking. Methods: Single-cell RNA sequencing data was analyzed to determine expression of m5C methylase NSUN2. m5C levels were determined by mRNA isolation and anti-m5C dot blot in both hypoxia-induced endothelial cells (ECs) and laser-induced choroidal neovascularization (CNV). In addition, endothelial cell and endothelium‐specific NSUN2‐knockout mouse model were used to investigate the effect of NSUN2 silence on angiogenic phenotype. Genome-wide multiomics analyses were performed to identify the functional target of NSUN2, including proteomic analysis, transcriptome screening and m5C-methylated RNA immunoprecipitation sequencing (m5C-meRIP-seq). CUT&Tag sequencing was performed to test the histone lactylation signal in the promoter region of NSUN2. Finally, AAV-mediated short hairpin RNAi knockdown of NSUN2 gene expression (AAV-shNSUN2) was constructed to investigate the effect of inhibiting CNV. Results: First, we discovered that m5C methylase NSUN2 expression level and mRNA m5C level were significantly higher in CNV-ECs than in normal ECs. NSUN2 knockdown in ECs inhibited proliferative, migration, and tube formation activities of ECs. Moreover, compared with EC NSUN2flox/flox mice, EC-specific NSUN2-deficient (EC NSUN2-/-) mice displayed less retinal vascular leakage after laser induction. Through multiomics analyses, we subsequently identified A-kinase anchoring protein 2 (AKAP2), a scaffolding protein which isolate Protein kinase A (PKA) to specific subcellular locations through binding to its regulatory subunit, as a downstream candidate target of NSUN2 in ECs. Overexpression of exogenous AKAP2 markedly reversed the inhibitory phenotypes in NSUN2-deficient ECs. Interestingly, laser induced NSUN2 up-regulation was driven by lactate-mediated lactylation on histone H3K18. In CNV models, AAV-mediated repression of NSUN2 modulated highly retinal vascular leakage and choroidal thickness. Conclusion: Overall, our findings indicate that NSUN2 is a novel therapeutic target for choroidal neovascularization.

Dataset Information

Identification of direct targets and modified bases of RNA cytosine methyltransferases

Publications

Identification of direct targets and modified bases of RNA cytosine methyltransferases.

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