Biology subjects

Toolan-Kerr, P.

Publications and source records attributed to Toolan-Kerr, P..

4 recordsLinked to original sources

bioRxiv2026

H3K37me1 couples transcription with DNA replication origin selection

DNA replication initiation in higher eukaryotes occurs at thousands of sites distributed throughout the genome and follows a defined temporal program. However, replication origins are not determined by a conserved DNA motif, and how specific genomic regions are selected for initiation remains poorly understood. Here, we identify a transcription-associated histone modification in human cells, mono-methylation of histone H3 lysine 37 (H3K37me1), and show that it regulates the spatial organization of replication initiation. H3K37me1 is enriched across actively transcribed gene bodies, and its depletion leads to a redistribution of replication origin activity toward intragenic regions. We show that H3K37me1 limits the association of the MCM2-7 replicative helicase with transcribed chromatin, thereby restricting intragenic origin usage. Consistently, loss of H3K37me1 increases both MCM2-7 occupancy and replication initiation across gene bodies. Collectively, our findings uncover a chromatin-based mechanism that couples transcription with DNA replication origin selection by limiting unscheduled origin firing within transcribed regions.

Molecular Biology↗

bioRxiv2026

BCLAF1 links RNA splicing to ATF4-dependent metabolic adaptation in acute myeloid leukemia

Acute myeloid leukemia (AML) is driven by a combination of genetic alterations and non-mutational mechanisms that disrupt normal hematopoiesis and support leukemic cell survival. While the mutational landscape of AML is well characterized, the non-genetic processes that sustain leukemic maintenance remain comparatively less understood. Using human AML cell lines and murine models of AML, we identify BCL2-associated transcription factor 1 (BCLAF1) as a key regulator of leukemic progression through control of mRNA processing. BCLAF1 physically associates with core spliceosome components and regulates alternative splicing, with a predominant effect on intron retention. We demonstrate that BCLAF1 is required for the productive splicing of activating transcription factor 4 (ATF4) mRNA, thereby sustaining ATF4 protein expression. Loss of BCLAF1 reduces ATF4 protein levels, leading to downregulation of metabolic target genes and disruption of de novo amino acid biosynthesis. Furthermore, depletion of BCLAF1 sensitizes AML cells to venetoclax, a clinically relevant BCL-2 inhibitor. Together, these findings uncover a previously unrecognized role for BCLAF1 in coordinating mRNA splicing and metabolic adaptation in AML, highlighting its potential as a therapeutic target. Statement of significanceAberrant RNA splicing and metabolic reprogramming are hallmarks of cancer, yet how these processes are mechanistically linked remains unclear. This study identifies BCLAF1 as a key regulator connecting splicing control to amino acid metabolism in acute myeloid leukemia, revealing a previously unrecognized functional vulnerability at the intersection of these pathways.

cancer biology↗

bioRxiv2022

m6A-ELISA, a simple method for quantifying N6-methyladenosine from mRNA populations

N6-methyladenosine (m6A) is a widely studied and abundant RNA modification. The m6A mark regulates the fate of RNAs in various ways, which in turn, drives changes in cell physiology, development, and disease pathology. Over the last decade, numerous methods have been developed to map and quantify m6A sites genomewide through deep sequencing. Alternatively, m6A levels can be quantified from a population of RNAs using techniques such as liquid chromatography-mass spectrometry or thin layer chromatography. However, many methods for quantifying m6A levels involve extensive protocols and specialized data analysis, and often only a few samples can be handled in a single experiment. Here, we developed a simple method for determining m6A levels in mRNA populations from various sources based on enzyme-linked immunosorbent-based assay (m6A-ELISA). We have optimized various steps of m6A-ELISA such as sample preparation and the background signal resulting from the primary antibody. We validated the method using mRNA populations from budding yeast and mouse embryonic stem cells. The full protocol takes less than a day, requiring only 25 ng of mRNA. The m6A-ELISA protocol is therefore quick, cost-effective, and scalable, making it a valuable tool for determining relative m6A levels in samples from various sources that could be adapted to detect other mRNA modifications.

biochemistry↗

bioRxiv2021

An improved iCLIP protocol

Crosslinking and Immunoprecipitation (CLIP) is a powerful technique to obtain transcriptome-wide maps of in vivo protein-RNA interactions, which are important to understand the post-transcriptional mechanisms mediated by RNA binding proteins (RBPs). Many variant CLIP protocols have been developed to improve the efficiency and convenience of cDNA library preparation. Here we describe an improved individual nucleotide resolution CLIP protocol (iiCLIP), which can be completed within 4 days from UV crosslinking to libraries for sequencing. For benchmarking, we directly compared PTBP1 iiCLIP libraries with the iCLIP2 protocol produced under standardised conditions, and with public eCLIP and iCLIP PTBP1 data. We visualised enriched motifs surrounding the identified crosslink positions and RNA maps of these crosslinks around the alternative exons regulated by PTBP1. Notably, motif enrichment was higher in iiCLIP and iCLIP2 in comparison to public eCLIP and iCLIP, and we show how this impacts the specificity of RNA maps. In conclusion, iiCLIP is technically convenient and efficient, and enables production of highly specific datasets for identifying RBP binding sites.

molecular biology↗