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Tag-Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases.

ABSTRACT: The mRNA modification N⁶ -methyladenosine (m⁶ A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3-METTL14 and METTL16 act as "writers" for different target transcripts and sequence motifs. The modification is perceived by dedicated "reader" and "eraser" proteins, but not by polymerases. We report that METTL3-14 shows remarkable cosubstrate promiscuity, enabling sequence-specific internal labeling of RNA without additional guide RNAs. The transfer of ortho-nitrobenzyl and 6-nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase-catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo-enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo-enzymatic modification of different sites in a single RNA.

SUBMITTER: Ovcharenko A

PROVIDER: S-EPMC7116812 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Tag-Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases.

Ovcharenko Anna A Weissenboeck Florian P FP Rentmeister Andrea A

Angewandte Chemie (International ed. in English) 20201222 8

The mRNA modification N<sup>6</sup> -methyladenosine (m<sup>6</sup> A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3-METTL14 and METTL16 act as "writers" for different target transcripts and sequence motifs. The modification is perceived by dedicated "reader" and "eraser" proteins, but not by polymerases. We report that METTL3-14 shows remarkable cosubstrate promiscuity, enabling sequence-specific internal labeling of RNA without additional guide R ...[more]

PMID: 33095964

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Tag-Free Internal RNA Labeling and Photocaging Based on mRNA Methyltransferases.

Project description:The mRNA modification N6 -methyladenosine (m6 A) is associated with multiple roles in cell function and disease. The methyltransferases METTL3-METTL14 and METTL16 act as "writers" for different target transcripts and sequence motifs. The modification is perceived by dedicated "reader" and "eraser" proteins, but not by polymerases. We report that METTL3-14 shows remarkable cosubstrate promiscuity, enabling sequence-specific internal labeling of RNA without additional guide RNAs. The transfer of ortho-nitrobenzyl and 6-nitropiperonyl groups allowed enzymatic photocaging of RNA in the consensus motif, which impaired polymerase-catalyzed primer extension in a reversible manner. METTL16 was less promiscuous but suitable for chemo-enzymatic labeling using different types of click chemistry. Since both enzymes act on distinct sequence motifs, their combination allowed orthogonal chemo-enzymatic modification of different sites in a single RNA.

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Visible-Light Removable Photocaging Groups Accepted by MjMAT Variant: Structural Basis and Compatibility with DNA and RNA Methyltransferases.

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Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing.

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Determining mRNA Stability by Metabolic RNA Labeling and Chemical Nucleoside Conversion.

Project description:The varying rates at which mRNAs decay are tightly coordinated with transcriptional changes to shape gene expression during development and disease. But currently available RNA sequencing approaches lack the temporal information to determine the relative contribution of RNA biogenesis, processing and turnover to the establishment of steady-state gene expression profiles.Here, we describe a protocol that combines metabolic RNA labeling with chemical nucleoside conversion by thiol-linked alkylation of 4-thiouridine to determine RNA stability in cultured cells (SLAMseq). When coupled to cost-effective mRNA 3' end sequencing approaches, SLAMseq determines the half-life of polyadenylated transcripts in a global and transcript-specific manner using untargeted or targeted cDNA library preparation protocols.We provide a step-by-step instruction for time-resolved mRNA 3' end sequencing, which augments traditional RNA-seq approaches to acquire the temporal resolution necessary to study the molecular principles that control gene expression.

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Dual-Functional-Tag-Facilitated Protein Labeling and Immobilization.

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Site-specific terminal and internal labeling of RNA by poly(A) polymerase tailing and copper-catalyzed or copper-free strain-promoted click chemistry.

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