Biology subjects

Lalonde, M.

Publications and source records attributed to Lalonde, M..

5 recordsLinked to original sources

bioRxiv2026

Aquarius RNA helicase Protects Pluripotent Stem Cell Identity

Pluripotent stem cells must reconcile rapid replication with a highly dynamic transcriptional program, creating an inherent susceptibility to transcription-replication conflicts (TRCs). We demonstrate that embryonic stem cells (ESCs) operate in a "resilient" replication mode, tolerating high genomic traffic through the constitutive upregulation of R-loop and TRC resolution pathways. Through a targeted functional screen, we identify the RNA helicase Aquarius (AQR) as an essential safeguard of this state. AQR depletion downregulates these resolution factors, collapsing this stress-resistant program and driving ESCs into unstable, heterogenous states, marked by increased transcriptional entropy and cell-to-cell noise. Mechanistically, we show that key identity-defining genes are preferentially located within R-loop and TRC prone regions, making them uniquely vulnerable to AQR depletion. Our findings establish AQR as a critical governor of transcriptional fidelity, demonstrating that genomic resilience is fundamental to maintaining pluripotent cell identity.

cell biology↗

bioRxiv2025

Proximity of pre-mRNA 3' end processing and transcription termination predicts enhanced gene expression

Two key factors required for pre-mRNA 3 end cleavage and polyadenylation, CPSF73 and PCF11, exhibit oncogenic properties: their elevated expression is associated with poor cancer patient prognosis. However, both proteins are also transcription termination factors, and it is unclear which of the processes they promote might contribute to carcinogenesis. Here, we employ a cellular model of colorectal cancer (CRC) progression and find that cells from primary tumor are addicted to high levels of CPSF73 and PCF11, while metastatic cells become less sensitive to their levels. We find no association between alternative polyadenylation (APA) and cell dependence on CPSF73 and PCF11, and no impact of their downregulation on transcription-replication collisions. Instead, we uncover an uncoupling of changes in 3 cleavage and termination during CRC progression: primary tumor-derived cells display a global shift to more proximal termination, yet a tendency for distal APA, compared to normal cells. Metastatic cells display partial reversion toward termination patterns observed in normal cells, and opposite tendency favoring proximal APA. This prompts us to measure the distance between the site of 3 cleavage and transcription termination for active protein-coding genes and find it almost halved in cells from primary tumor compared to normal cells. Interestingly, this distance becomes critically short for oncogenes. Closer proximity of 3 cleavage to termination correlates with higher gene expression, both across genes within a cell line and when distance and expression change in parallel. This uncovers a new relationship of transcription termination with gene expression regulation. SIGNIFICANCE STATEMENTAt the 3 end of genes two processes occur, which are often treated as one: pre-mRNA 3 processing and transcription termination. In fact, they are promoted by a common set of protein factors, with oncogenic properties. Here, we employ a cellular model of colorectal cancer (CRC) progression, to study changes in both pre-mRNA 3 processing and transcription termination. Unexpectedly, we uncover that those changes occur in opposite directions: mRNA tends to be lengthened, while termination is accelerated in cells from primary tumors. We show that the cleavage-termination distance is strongly reduced in these cells compared to normal and metastatic cells, and further determine that cleavage-termination proximity correlates with enhanced gene expression. This uncovers a new layer of gene expression regulation.

molecular biology↗

bioRxiv2025

Replisome progression regulates R-loop mediated transcriptional repression

Maintaining cellular proliferation necessitates the synchronized activity of diverse molecular machineries operating in parallel on the genome. Wide-spread transcription and R-loop formation can interfere with genome duplication, causing transcription-replication conflicts (TRCs). Here, we use live-cell imaging for simultaneous monitoring of replication fork progression and transcription dynamics of an R-loop prone gene. While robust replisome progression through R-loops is observed in wild-type cells, it is impaired in RnaseH, Mph1 and Sen1 mutants. Intriguingly, we find that R-loop formation inhibits gene transcription, but this inhibition is reversed by replisome passage, demonstrating the dynamic crosstalk between replication, transcription and R-loops in a single cell cycle. Unexpectedly, we find that R-loops also have beneficial roles in reducing the density of RNAPII molecules on the gene, thereby preventing fork stalling upon high RNAPII occupancy. These findings illuminate that regulating R-loops and RNAPII density together is critical for preventing harmful TRCs.

cell biology↗

bioRxiv2023

Single molecule MATAC-seq reveals key determinants of DNA replication origin efficiency

SummaryStochastic origin activation gives rise to significant cell-to-cell variability in the pattern of genome replication. The molecular basis for heterogeneity in efficiency and timing of individual origins is a long-standing question. Here, we developed Methylation Accessibility of TArgeted Chromatin domain Sequencing (MATAC-Seq) to determine single-molecule chromatin accessibility of specific genomic loci after targeted purification in their native chromatin context. Applying MATAC-Seq to selected early-efficient (EE) and late-inefficient (LI) budding yeast replication origins revealed large heterogeneity of chromatin states. Disruption of INO80 or ISW2 chromatin remodeling complexes leads to changes at individual nucleosomal positions that correlate with changes in their replication efficiency. We found a chromatin state with an optimal 100-115bp nucleosome-free region in combination with surrounding well-positioned nucleosomes and open +2 linker region is a strong predictor for efficient origin activation. Thus, MATAC-Seq identifies the large spectrum of alternative chromatin states that co-exist on a given locus previously masked in population-based experiments and provides a mechanistic basis for origin activation heterogeneity during DNA replication of eukaryotic cells. Consequently, our single-molecule assay for chromatin accessibility will be ideal to define single-molecule heterogeneity across many fundamental biological processes such as transcription, replication, or DNA repair in vitro and ex vivo.

biochemistry↗

bioRxiv2023

The CGG triplet repeat binding protein 1 counteracts DNA secondary structure-induced transcription-replication conflicts

Short tracts of trinucleotide repeats with less than 10 repeats are found frequently throughout the genome without any apparent negative impact on DNA replication fork progression or transcription elongation. CGG binding protein 1 (CGGBP1) binds to CGG triplet repeats and has been implicated in multiple cellular processes such as transcription, replication and DNA damage. Here, we show that CGGBP1 binds to human gene promoter sites prone to G-quadruplex and R-loop secondary structure formation. Altering CGGBP1 levels results in the accumulation of R-loops and causes a defect in transcriptional elongation by RNA polymerase II, which subsequently leads to replication fork stalling and transcription-replication conflicts. Together, our work shows that short trinucleotide repeats are a source of genome-destabilizing secondary structures and cells rely on specific DNA-binding factors to maintain proper transcription and replication progression at short trinucleotide repeats.

cell biology↗