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5‐Formylcytosine (f5C) modification is present in human mitochondrial methionine tRNA (mt‐tRNAMet) and cytosolic leucine tRNA (ct‐tRNALeu), with their formation mediated by NSUN3 and ALKBH1. f5C has also been detected in yeast mRNA and human tRNA, but its transcriptome‐wide distribution in mammals has not been studied. Here we report f5C‐seq, a quantitative sequencing method to map f5C transcriptome‐wide in HeLa and mouse embryonic stem cells (mESCs). We show that f5C in RNA can be reduced to dihydrouracil (DHU) by pic‐borane, and DHU can be exclusively read as T during reverse transcription (RT) reaction, allowing the detection and quantification of f5C sites by a unique C‐to‐T mutation signature. We validated f5C‐seq by identifying and quantifying the two known f5C sites in tRNA, in which the f5C modification fractions dropped significantly in ALKBH1‐depleted cells. By applying f5C‐seq to chromatin‐associated RNA (caRNA), we identified several highly modified f5C sites in HeLa and mouse embryonic stem cells (mESC). [ABSTRACT FROM AUTHOR]

Motivation Kethoxal-assisted ssDNA sequencing (KAS-seq) is rapidly gaining popularity as a robust and effective approach to study the nascent dynamics of transcriptionally engaged RNA polymerases through profiling of genome-wide single-stranded DNA (ssDNA). Its latest variant, spKAS-seq, a strand-specific version of KAS-seq, has been developed to map genome-wide R-loop structures by detecting imbalances of ssDNA on two strands. However, user-friendly, open-source computational tools tailored for KAS-seq data are still lacking. Results Here, we introduce KAS-Analyzer, the first comprehensive computational framework aimed at streamlining and enhancing the analysis and interpretation of KAS-seq and spKAS-seq data. In addition to standard analyses, KAS-Analyzer offers many novel tools specifically designed for KAS-seq data, including, but not limited to: calculation of transcription-related metrics, identification of single-stranded transcribing (SST) enhancers, high-resolution mapping of R-loops, and differential RNA polymerase activity analysis. We provided a detailed overview of KAS-seq data and its diverse applications through the implementation of KAS-Analyzer. Using the example time-course KAS-seq datasets, we further showcase the robust capabilities of KAS-Analyzer for investigating dynamic transcriptional regulatory programs in response to UVB radiation. Availability and implementation KAS-Analyzer is available at https://github.com/Ruitulyu/KAS-Analyzer. [ABSTRACT FROM AUTHOR]

N 1 -methyl adenosine (m 1 A) is a widespread RNA modification present in tRNA, rRNA, and mRNA. m 1 A modification sites in tRNAs are evolutionarily conserved and its formation on tRNA is catalyzed by methyltransferase TRMT61A and TRMT6 complex. m 1 A promotes translation initiation and elongation. Due to its positive charge under physiological conditions, m 1 A can notably modulate RNA structure. It also blocks Watson-Crick-Franklin base-pairing and causes mutation and truncation during reverse transcription. Several misincorporation-based high-throughput sequencing methods have been developed to sequence m 1 A. In this study, we introduce a reduction-based m 1 A sequencing (red-m 1 A-seq). We report that NaBH 4 reduction of m 1 A can improve the mutation and readthrough rates using commercially available RT enzymes to give a better positive signature, while alkaline-catalyzed Dimroth rearrangement can efficiently convert m 1 A to m 6 A to provide good controls, allowing the detection of m 1 A with higher sensitivity and accuracy. We applied red-m 1 A-seq to sequence human small RNA, and we not only detected all the previously reported tRNA m 1 A sites, but also new m 1 A sites in mt-tRNA Asn-GTT and 5.8S rRNA., (© 2024 Pajdzik et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

The RNA N 6 -methyladenosine (m 6 A) methylation is installed by the METTL3-METTL14 methyltransferase complex. This modification has critical regulatory roles in various biological processes. Here, we report that deletion of Mettl14 dramatically reduces mRNA m 6 A methylation in developing B cells and severely blocks B cell development in mice. Deletion of Mettl14 impairs interleukin-7 (IL-7)-induced pro-B cell proliferation and the large-pre-B-to-small-pre-B transition and causes dramatic abnormalities in gene expression programs important for B cell development. Suppression of a group of transcripts by cytoplasmic m 6 A reader YTHDF2 is critical to the IL-7-induced pro-B cell proliferation. In contrast, the block in the large-pre-B-to-small-pre-B transition is independent of YTHDF1 or YTHDF2 but is associated with a failure to properly upregulate key transcription factors regulating this transition. Our data highlight the important regulatory roles of the RNA m 6 A methylation and its reader proteins in early B cell development., Competing Interests: Declaration of Interests C.H. is a scientific founder and a scientific advisory board member of Accent Therapeutic, Inc. H.H. is an employee of Genentech., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

N 6 -methyladenosine (m 6 A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m 6 A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m 6 A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m 6 A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m 6 A-mediated splicing regulation, in which an m 6 A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m 6 A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing., (Copyright © 2019 Elsevier Inc. All rights reserved.)