Biology subjects

Proudfoot, N. J.

Publications and source records attributed to Proudfoot, N. J..

7 recordsLinked to original sources

bioRxiv2026

Nucleolar endonuclease MBLAC2 orchestrates co-transcriptional ribosomal RNA processing and global transcriptional output

The metallo-{beta}-lactamase (MBL)-domain superfamily includes multiple nucleases that process distinct transcript classes. We show that human MBLAC2 acts as a ribosomal (r)RNA quality control factor, being nucleolar specific, chromatin-associated and required for co-transcriptional pre-rRNA processing. Notably MBLAC2 is functionally distinct from other MBL containing proteins, being required for maintenance of nucleolar architecture, cell cycle progression and proliferative fitness. Selective MBLAC2 depletion causes accumulation of pre-rRNA intermediates that lead to perturbed RNA polymerase (Pol) I-associated RNA homeostasis and nucleolar stress signalling. Nascent RNA profiling with long-read nanopore sequencing reveals extended and aberrant pre-rRNA species, consistent with defective processing and transcript release. Remarkably disruption of rRNA processing reduces global Pol II transcriptional output, even though the Pol II nuclear distribution remains unchanged across genomic compartments. Altogether, we define MBLAC2 as the nucleolar branch of an MBL-domain RNA surveillance network that couples rRNA quality control with transcriptional homeostasis.

molecular biology↗

bioRxiv2025

An SMN2 splicing-correcting antisense oligonucleotide promotes gene looping

We have previously proposed a combined therapy for spinal muscular atrophy based on the upregulation of exon 7 (E7) inclusion into SMN2 mRNA by the antisense oligonucleotide nusinersen (ASO1) together with histone deacetylase inhibitors (HDACi). Using a CRISPR/deadCas-based strategy, we show here that targeting histone acetylation at the SMN2 E7 region duplicates the HDACi effect, which overcomes pleiotropic effects. Most noticeably, targeted acetylation at the E7 region causes acetylation and activation of the 30-kbp-distant promoter. Since E7 and the nusinersen target site map near the SMN2 gene 3'-end, this cross-talk between the two ends of the gene prompted us to use chromosome conformation analysis (3C) to find that ASO1 promotes gene looping. This unpredicted property of ASOs in modulating chromatin 3D configuration depends on cohesin, CTCF, cleavage/polyadenylation factors and a canonical poly(A) signal, and causes promoter activation at a long distance, which expands the therapeutic potential of splicing-correcting ASOs.

molecular biology↗

bioRxiv2024

DNA-directed termination of mammalian RNA polymerase II

The best-studied mechanism of eukaryotic RNA polymerase II (RNAPII) transcriptional termination involves polyadenylation site-directed cleavage of the nascent RNA. The RNAPII-associated cleavage product is then degraded by XRN2, dislodging RNAPII from the DNA template. In contrast, prokaryotic RNAP and eukaryotic RNAPIII often terminate directly at T-tracts in the coding DNA strand. Here, we demonstrate a similar and omnipresent capability for mammalian RNAPII. XRN2- and T-tract-dependent termination are independent - the latter usually acting when XRN2 cannot be engaged. We show that T-tracts terminate snRNA transcription, previously thought to require the Integrator complex. Importantly, we find genome-wide termination at T-tracts in promoter-proximal regions, but not within protein-coding gene bodies. XRN2-dependent termination dominates downstream of protein-coding genes, but the T-tract process is sometimes employed. Overall, we demonstrate global DNA-directed attrition of RNAPII transcription, suggesting that RNAPs retain the potential to terminate over T-rich sequences throughout evolution.

molecular biology↗

bioRxiv2023

Pro-inflammatory polarization and colorectal cancer modulate alternative and intronic polyadenylation in primary human macrophages

Macrophages are essential cells of the immune system that alter their inflammatory profile depending on their microenvironment. Alternative polyadenylation in the 3UTR (3UTR-APA) and intronic polyadenylation (IPA) are mechanisms that modulate gene expression, in particular in cancer and activated immune cells. Yet, how polarization and colorectal cancer (CRC) cells microenvironment affect 3UTR-APA and IPA in primary human macrophages remains unknown. Here, primary human monocytes were isolated from healthy donors, differentiated and polarized into a pro-inflammatory state and ChrRNA-Seq and 3RNA-Seq were performed to quantify gene expression and characterize new 3UTR-APA and IPA mRNA isoforms. Our results show that polarization of human macrophages from naive to a pro-inflammatory state causes a marked increase both in proximal polyA site selection in the 3UTR and in IPA events, in genes relevant for macrophage functions. Additionally, we found a negative correlation between differential gene expression and IPA during pro-inflammatory polarization of primary human macrophages. As macrophages are abundant immune cells in the CRC microenvironment that either promote or abrogate cancer progression, we investigated how indirect exposure to CRC cells affects macrophage gene expression and 3UTR-APA and IPA mRNA events. Co-culture with CRC cells alters the inflammatory phenotype of macrophages, increases the expression of pro-tumoral genes and induce 3UTR-APA alterations. Notably, some of these gene expression differences were also found in tumour-associated macrophages of CRC patients, indicating that they are physiological relevant. Upon macrophage pro-inflammatory polarization SRSF12 is the pre-mRNA processing gene that is most upregulated. After SRSF12 knockdown in M1 macrophages there is a global downregulation of gene expression, in particular in genes involved in gene expression regulation and in immune responses. Our results reveal new 3UTR-APA and IPA mRNA isoforms produced during pro-inflammatory polarization of primary human macrophages and CRC co-culture that may be used in the future as diagnostic or therapeutic tools.

molecular biology↗

bioRxiv2019

Enhancers predominantly regulate gene expression in vivo via transcription initiation.

Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

genomics↗

bioRxiv2018

Selective roles of vertebrate PCF11 in premature and full-length transcript termination

The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including: mNET-seq, 3 mRNA-seq, chromatin RNA-seq and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and downstream gene silencing. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript, and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.

molecular biology↗

bioRxiv2018

S phase R-loop formation is restricted by PrimPol-mediated repriming

During DNA replication, conflicts with ongoing transcription are frequent and require careful management to avoid genetic instability. R-loops, three stranded nucleic acid structures comprising a DNA:RNA hybrid and displaced single stranded DNA, are important drivers of damage arising from such conflicts. How R-loops stall replication and the mechanisms that restrain their formation during S phase are incompletely understood. Here we show in vivo how R-loop formation drives a short purine-rich repeat, (GAA)10, to become a replication impediment that requires the repriming activity of the primase-polymerase PrimPol for its processive replication. Further, we show that loss of PrimPol results in a significant increase in R-loop formation around the repeat during S phase. We extend this observation by showing that PrimPol suppresses R-loop formation in genes harbouring secondary structure-forming sequences, exemplified by G quadruplex and H-DNA motifs, across the genome in both avian and human cells. Thus, R-loops promote the creation of replication blocks at susceptible sequences, while PrimPol-dependent repriming limits the extent of unscheduled R-loop formation at these sequences, mitigating their impact on replication.

molecular biology↗