Post-transcriptional polyadenylation site cleavage maintains 3'-end processing upon DNA damage.

The recognition of polyadenylation signals (PAS) in eukaryotic pre-mRNAs is usually coupled to transcription termination, occurring while pre-mRNA is chromatin-bound. However, for some pre-mRNAs, this 3'-end processing occurs post-transcriptionally, i.e., through a co-transcriptional cleavage (CoTC) event downstream of the PAS, leading to chromatin release and subsequent PAS cleavage in the nucleoplasm. While DNA-damaging agents trigger the shutdown of co-transcriptional chromatin-associated 3'-end processing, specific compensatory mechanisms exist to ensure efficient 3'-end processing for certain pre-mRNAs, including those that encode proteins involved in the DNA damage response, such as the tumor suppressor p53. We show that cleavage at the p53 polyadenylation site occurs in part post-transcriptionally following a co-transcriptional cleavage event. Cells with an engineered deletion of the p53 CoTC site exhibit impaired p53 3'-end processing, decreased mRNA and protein levels of p53 and its transcriptional target p21, and altered cell cycle progression upon UV-induced DNA damage. Using a transcriptome-wide analysis of PAS cleavage, we identify additional pre-mRNAs whose PAS cleavage is maintained in response to UV irradiation and occurring post-transcriptionally. These findings indicate that CoTC-type cleavage of pre-mRNAs, followed by PAS cleavage in the nucleoplasm, allows certain pre-mRNAs to escape 3'-end processing inhibition in response to UV-induced DNA damage.

At the crossroads of RNA biology, genome integrity and cancer.

This article is the synthesis of the scientific presentations that took place during two international courses at Institute Curie, one on post-transcriptional gene regulation and the other on genome instability and human disease, that were joined together in their 2021 edition. This joined course brought together the knowledge on RNA metabolism and the maintenance of genome stability.

Differential Effects on the Translation of Immune-Related Alternatively Polyadenylated mRNAs in Melanoma and T Cells by eIF4A Inhibition.

Targeting the translation initiation complex eIF4F, which binds the 5' cap of mRNAs, is a promising anti-cancer approach. Silvestrol, a small molecule inhibitor of eIF4A, the RNA helicase component of eIF4F, inhibits the translation of the mRNA encoding the signal transducer and activator of transcription 1 (STAT1) transcription factor, which, in turn, reduces the transcription of the gene encoding one of the major immune checkpoint proteins, i.e., programmed death ligand-1 (PD-L1) in melanoma cells. A large proportion of human genes produce multiple mRNAs differing in their 3'-ends through the use of alternative polyadenylation (APA) sites, which, when located in alternative last exons, can generate protein isoforms, as in the STAT1 gene. Here, we provide evidence that the STAT1α, but not STAT1ß protein isoform generated by APA, is required for silvestrol-dependent inhibition of PD-L1 expression in interferon-γ-treated melanoma cells. Using polysome profiling in activated T cells we find that, beyond STAT1, eIF4A inhibition downregulates the translation of some important immune-related mRNAs, such as the ones encoding TIM-3, LAG-3, IDO1, CD27 or CD137, but with little effect on the ones for BTLA and ADAR-1 and no effect on the ones encoding CTLA-4, PD-1 and CD40-L. We next apply RT-qPCR and 3'-seq (RNA-seq focused on mRNA 3' ends) on polysomal RNAs to analyze in a high throughput manner the effect of eIF4A inhibition on the translation of APA isoforms. We identify about 150 genes, including TIM-3, LAG-3, AHNAK and SEMA4D, for which silvestrol differentially inhibits the translation of APA isoforms in T cells. It is therefore crucial to consider 3'-end mRNA heterogeneity in the understanding of the anti-tumor activities of eIF4A inhibitors.

Salmonella Typhimurium Infection Leads to Colonization of the Mouse Brain and Is Not Completely Cured With Antibiotics.

Salmonella systemic infections claim thousands of lives worldwide even today. Certain cases lead to an infection in the brain culminating in meningitis and associated neurological abnormalities. Multiple reports have indicated neurological manifestations in patients suffering from typhoid fever during the course of infection and afterwards. While the meanderings of Salmonella systemic infections are fairly well studied, the flow of events in the brain is very poorly understood. We investigated the colonization of various brain parts by Salmonella in mice. It was observed that the bacterium is frequently able to invade various brain parts in mice. Selected mutants namely deletion mutants of key proteins encoded by the Salmonella pathogenicity islands (SPIs) 1 and 2 and ompA gene were also used to decipher the roles of specific genes in establishing an infection in the brain. Our results suggest roles for the Salmonella pathogenicity island (SPI) 1 and outer membrane protein A gene in enabling blood-brain barrier penetration by the pathogen. We further investigated behavioral abnormalities in infected mice and used an antibiotic treatment regime in an attempt to reverse the same. Results show some mice still display behavioral abnormalities and a high bacterial burden in brain despite clearance from spleen and liver. Overall, our study provides novel insights into S. Typhimurium's capacity to invade the mouse brain and the effectiveness of antibiotic treatment on behavioral manifestations due to infection. These observations could have important implications in understanding reported neurological manifestations in typhoid patients.