Biology subjects

Saponaro, M.

Publications and source records attributed to Saponaro, M..

6 recordsLinked to original sources

bioRxiv2025

MYBL2 maintains stemness and promotes theta-mediated end joining in triple negative breast cancer

Triple negative breast cancer (TNBC) has a poor prognosis due to limited treatment options and high metastasis risk. Breast cancer (BC) stem/progenitor cells, also known as tumour initiating cells, are a small, difficult to target population within the tumour responsible for metastasis and therapy resistance. MYBL2 overexpression is commonly linked to poor prognosis and metastasis but its role in BC stem/progenitor cells remains elusive. To determine the role of MYBL2 in BC stem/progenitor cells we generated TNBC cell lines with inducible MYBL2 downregulation. Our findings reveal that elevated MYBL2 is essential for self-renewal, DNA repair, and replication stress response in these cells and lowering MYBL2 impairs stemness and self-renewal both in vitro and in vivo. Accordingly, our functional and mechanistic analyses indicate that high-MYBL2 stem/progenitor cells exhibit increased sensitivity to Pol{theta} inhibitors which is lost upon MYBL2 downregulation due to transcriptional suppression. Combining Pol{theta} inhibitors with ATR inhibitors further enhances this sensitivity, in vitro and ex vivo. This study thus identifies Pol{theta}/ATR inhibition as a synthetic lethality strategy to eradicate BC stem/progenitor cells and underscores MYBL2 expression as a biomarker for patient stratification in this treatment.

cancer biology↗

bioRxiv2024

RNA Polymerase II subunits overexpressions induce genome instability and deregulate transcription

Independently of the pathways or circuits deregulated in cancer cells, these present altered transcription patterns, often also direct consequence of deregulation of transcription factors. In this sense, also the RNA Polymerase complexes responsible for transcription can be affected in cancers. We find that upregulations of RNA Polymerase II subunits, especially the largest ones, correlates with poor cancer patients outcome across a range of tumor types, presenting increased genome instability. Overexpressing the subunits RPB1, RPB3 and RPB4 in cells we find that these induce DNA damage. However, the mechanisms behind this increased genome instability are specific for each subunit, linked to the unique transcription alterations generated by the subunit overexpression. Importantly, we find significant overlap between the genes with more DNA damage in our cell line models and those more affected in cancers with subunit upregulation, indicating that upregulations could be responsible for some of the phenotypes present in these patients.

molecular biology↗

bioRxiv2024

USP50 suppresses alternative RecQ helicase use and deleterious DNA2 activity during replication.

Mammalian DNA replication employs several RecQ DNA helicases to orchestrate the faithful duplication of genetic information. Helicase function is often coupled to the activity of specific nucleases, but how helicase and nuclease activities are co-directed is unclear. Here we identify the inactive ubiquitin-specific protease, USP50, as a ubiquitin-binding and chromatin-associated protein required for ongoing replication, fork restart, telomere maintenance and cellular survival during replicative stress. USP50 supports WRN:FEN1 at stalled replication forks, suppresses MUS81-dependent fork collapse and restricts double-strand DNA breaks at GC-rich sequences. Surprisingly we find that cells depleted for USP50 and recovering from a replication block exhibit increased DNA2 and RECQL4 foci and that the defects in ongoing replication, poor fork restart and increased fork collapse seen in these cells are mediated by DNA2, RECQL4 and RECQL5. These data define a novel ubiquitin-dependent pathway that promotes the balance of helicase: nuclease use at ongoing and stalled replication forks.

cancer biology↗

bioRxiv2022

Ubiquitin ligase TRAIP plays an essential role during the S-phase of unperturbed cell cycle in the resolution of DNA replication - transcription conflicts

Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, for the first time, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We find that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.

cell biology↗

bioRxiv2021

RPRD Proteins Control Transcription in Human Cells

The regulation of transcription is an essential process that allows the cell to respond to various internal and external signals. RNA Polymerase II (Pol II) activity is controlled by a number of factors which bind to the C-terminal domain (CTD) of its largest subunit, RPB1, and stimulate or suppress RNA synthesis. Here, we demonstrate that CTD-interacting proteins, RPRD2, RPRD1B and RPRD1A act as negative regulators of transcription and their levels inversely correlate with the accumulation of nascent and newly transcribed RNA in human cells. We show that the RPRD proteins form mutually exclusive complexes with Pol II to coordinate their roles in transcriptional control. Our data indicate that RPRD2 exerts the most substantial impact on transcription and has the potential to alter key biological processes including the cellular stress response and cell growth.

molecular biology↗

bioRxiv2021

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive and it correlates with Pol II transcription and mRNA expression levels. While changes in Pol II occupancy were detected at only some genes in a Upf1-deficient (upf1{Delta}) strain, there is an increased Ser2 Pol II signal at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1{Delta} cells are hypersensitive to the transcription elongation inhibitor 6-azauracil and display Pol II abnormalities suggestive of Pol II hyperphosphorylation. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1{Delta}. These data envisage that by operating on the nascent transcript Upf1 might influence Pol II phosphorylation and transcription.

molecular biology↗