DDX17 is required for efficient DSB repair at DNA:RNA hybrid deficient loci.

Proteins with RNA-binding activity are increasingly being implicated in DNA damage responses (DDR). Additionally, DNA:RNA-hybrids are rapidly generated around DNA double-strand breaks (DSBs), and are essential for effective repair. Here, using a meta-analysis of proteomic data, we identify novel DNA repair proteins and characterise a novel role for DDX17 in DNA repair. We found DDX17 to be required for both cell survival and DNA repair in response to numerous agents that induce DSBs. Analysis of DSB repair factor recruitment to damage sites suggested a role for DDX17 early in the DSB ubiquitin cascade. Genome-wide mapping of R-loops revealed that while DDX17 promotes the formation of DNA:RNA-hybrids around DSB sites, this role is specific to loci that have low levels of pre-existing hybrids. We propose that DDX17 facilitates DSB repair at loci that are inefficient at forming DNA:RNA-hybrids by catalysing the formation of DSB-induced hybrids, thereby allowing propagation of the damage response.

YTHDC2 control of gametogenesis requires helicase activity but not m6A binding.

Mechanisms regulating meiotic progression in mammals are poorly understood. The N6-methyladenosine (m6A) reader and 3' → 5' RNA helicase YTHDC2 switches cells from mitotic to meiotic gene expression programs and is essential for meiotic entry, but how this critical cell fate change is accomplished is unknown. Here, we provide insight into its mechanism and implicate YTHDC2 in having a broad role in gene regulation during multiple meiotic stages. Unexpectedly, mutation of the m6A-binding pocket of YTHDC2 had no detectable effect on gametogenesis and mouse fertility, suggesting that YTHDC2 function is m6A-independent. Supporting this conclusion, CLIP data defined YTHDC2-binding sites on mRNA as U-rich and UG-rich motif-containing regions within 3' UTRs and coding sequences, distinct from the sites that contain m6A during spermatogenesis. Complete loss of YTHDC2 during meiotic entry did not substantially alter translation of its mRNA binding targets in whole-testis ribosome profiling assays but did modestly affect their steady-state levels. Mutation of the ATPase motif in the helicase domain of YTHDC2 did not affect meiotic entry, but it blocked meiotic prophase I progression, causing sterility. Our findings inform a model in which YTHDC2 binds transcripts independent of m6A status and regulates gene expression during multiple stages of meiosis by distinct mechanisms.

The roles of RNA in DNA double-strand break repair.

Effective DNA repair is essential for cell survival: a failure to correctly repair damage leads to the accumulation of mutations and is the driving force for carcinogenesis. Multiple pathways have evolved to protect against both intrinsic and extrinsic genotoxic events, and recent developments have highlighted an unforeseen critical role for RNA in ensuring genome stability. It is currently unclear exactly how RNA molecules participate in the repair pathways, although many models have been proposed and it is possible that RNA acts in diverse ways to facilitate DNA repair. A number of well-documented DNA repair factors have been described to have RNA-binding capacities and, moreover, screens investigating DNA-damage repair mechanisms have identified RNA-binding proteins as a major group of novel factors involved in DNA repair. In this review, we integrate some of these datasets to identify commonalities that might highlight novel and interesting factors for future investigations. This emerging role for RNA opens up a new dimension in the field of DNA repair; we discuss its impact on our current understanding of DNA repair processes and consider how it might influence cancer progression.

The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112.

N6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5-TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5-TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid.

Identification of the m6Am Methyltransferase PCIF1 Reveals the Location and Functions of m6Am in the Transcriptome.

mRNAs are regulated by nucleotide modifications that influence their cellular fate. Two of the most abundant modified nucleotides are N6-methyladenosine (m6A), found within mRNAs, and N6,2'-O-dimethyladenosine (m6Am), which is found at the first transcribed nucleotide. Distinguishing these modifications in mapping studies has been difficult. Here, we identify and biochemically characterize PCIF1, the methyltransferase that generates m6Am. We find that PCIF1 binds and is dependent on the m7G cap. By depleting PCIF1, we generated transcriptome-wide maps that distinguish m6Am and m6A. We find that m6A and m6Am misannotations arise from mRNA isoforms with alternative transcription start sites (TSSs). These isoforms contain m6Am that maps to "internal" sites, increasing the likelihood of misannotation. We find that depleting PCIF1 does not substantially affect mRNA translation but is associated with reduced stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Am's function.

Transcriptome-Wide Mapping of m6 A and m6 Am at Single-Nucleotide Resolution Using miCLIP.

The most prevalent modified base in mRNA, N6 -methyladenosine (m6 A), is found in several thousand transcripts, typically near the stop codon, although it can occur anywhere in the mRNA. In addition, the highly similar nucleotide N6 ,2'-O-dimethyladenosine (m6 Am), which is difficult to distinguish from m6 A, occurs as the first transcribed nucleotide of certain transcripts. Both the m6 A and m6 Am modifications have been implicated in numerous biological processes, and their precise mapping is crucial to understanding their functions. To address this need, we developed miCLIP, a method that maps both m6 A and m6 Am at individual nucleotide resolution. Here we describe the miCLIP protocol, with slight improvements to the initially published protocol for both the experimental methodology and bioinformatics analysis. © 2019 by John Wiley & Sons, Inc.

FTO controls reversible m6Am RNA methylation during snRNA biogenesis.

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

Drosha drives the formation of DNA:RNA hybrids around DNA break sites to facilitate DNA repair.

The error-free and efficient repair of DNA double-stranded breaks (DSBs) is extremely important for cell survival. RNA has been implicated in the resolution of DNA damage but the mechanism remains poorly understood. Here, we show that miRNA biogenesis enzymes, Drosha and Dicer, control the recruitment of repair factors from multiple pathways to sites of damage. Depletion of Drosha significantly reduces DNA repair by both homologous recombination (HR) and non-homologous end joining (NHEJ). Drosha is required within minutes of break induction, suggesting a central and early role for RNA processing in DNA repair. Sequencing of DNA:RNA hybrids reveals RNA invasion around DNA break sites in a Drosha-dependent manner. Removal of the RNA component of these structures results in impaired repair. These results show how RNA can be a direct and critical mediator of DNA damage repair in human cells.

The emerging role of RNAs in DNA damage repair.

This corrects the article DOI: 10.1038/cdd.2017.16.

The emerging role of RNAs in DNA damage repair.

Many surveillance and repair mechanisms exist to maintain the integrity of our genome. All of the pathways described to date are controlled exclusively by proteins, which through their enzymatic activities identify breaks, propagate the damage signal, recruit further protein factors and ultimately resolve the break with little to no loss of genetic information. RNA is known to have an integral role in many cellular pathways, but, until very recently, was not considered to take part in the DNA repair process. Several reports demonstrated a conserved critical role for RNA-processing enzymes and RNA molecules in DNA repair, but the biogenesis of these damage-related RNAs and their mechanisms of action remain unknown. We will explore how these new findings challenge the idea of proteins being the sole participants in the response to DNA damage and reveal a new and exciting aspect of both DNA repair and RNA biology.