Dataset Information

Programmable m⁶A modification of cellular RNAs with a Cas13-directed methyltransferase.

ABSTRACT: N⁶-Methyladenosine (m⁶A) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of m⁶A, the inability to install m⁶A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m⁶A and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a truncated METTL3 methyltransferase domain and cytoplasm-localized fusions with a modified METTL3:METTL14 methyltransferase complex can direct site-specific m⁶A incorporation in distinct cellular compartments, with the former fusion protein having particularly low off-target activity. Independent cellular assays across multiple sites confirm that this targeted RNA methylation (TRM) system mediates efficient m⁶A installation in endogenous RNA transcripts with high specificity. Finally, we show that TRM can induce m⁶A-mediated changes to transcript abundance and alternative splicing. These findings establish TRM as a tool for targeted epitranscriptome engineering that can reveal the effect of individual m⁶A modifications and dissect their functional roles.

SUBMITTER: Wilson C

PROVIDER: S-EPMC7718427 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase.

Wilson Christopher C Chen Peter J PJ Miao Zhuang Z Liu David R DR

Nature biotechnology 20200629 12

N6-Methyladenosine (m6A) is the most widespread internal messenger RNA modification in humans. Despite recent progress in understanding the biological roles of m6A, the inability to install m6A site specifically in individual transcripts has hampered efforts to elucidate causal relationships between the presence of a specific m6A and phenotypic outcomes. In the present study, we demonstrate that nucleus-localized dCas13 fusions with a trunca ...[more]

PMID: 32601430

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Project description:BackgroundTargeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3' adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2'-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3' sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA.ResultsWe showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3' end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs.ConclusionsOur findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

Dataset Information

Programmable m⁶A modification of cellular RNAs with a Cas13-directed methyltransferase.

Publications

Programmable m<sup>6</sup>A modification of cellular RNAs with a Cas13-directed methyltransferase.

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