Biology subjects

Hawley, B. R.

Publications and source records attributed to Hawley, B. R..

3 recordsLinked to original sources

bioRxiv2024

Decoding m6Am by simultaneous transcription-start mapping and methylation quantification

N6,2-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5 isoforms. Thus, gene levels annotations cannot capture the diversity of m6Am modification in the transcriptome. Here we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5 isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.

molecular biology↗

bioRxiv2021

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 are naturally deficient for DNA:RNA-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.

cell biology↗

bioRxiv2018

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. A long-standing challenge has been distinguishing these similar modifications in transcriptome-wide mapping studies. 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 definitively identified m6Am sites and generated transcriptome-wide maps that are selective for m6Am and m6A. We find that m6A and m6Am misannotations largely arise from mRNA isoforms with alternate transcription-start sites. These isoforms contain m6Am that appear to map to \"internal\" sites, increasing the likelihood of misannotation. Using the new m6Am annotations, we find that depleting m6Am does not affect mRNA translation but reduces the stability of a subset of m6Am-annotated mRNAs. The discovery of PCIF1 and our accurate mapping technique will facilitate future studies to characterize m6Ams function.

molecular biology↗