mA RNA methylation controls salivary gland epithelial cell function and has a protective role in Sjögren's disease.

Objectives: The RNA epitranscriptomic modification known as -methyladenosine (mA) represents a novel mechanism of gene regulation that is poorly understood in human autoimmune diseases. Our research explores the role of this RNA mA modification in salivary gland epithelial cells (SGEC) and its impact on the pathogenesis of Sjögren's disease (SjD).

Methods: SGECs from SjD patients and controls were analysed for mA writers METTL3 and METTL14 expression using RNA-seq, quantitative PCR and immunohistochemistry.

Protocol to study the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using cross-linking immunoprecipitation.

RNA-binding proteins (RBPs) are involved in many biological processes. The direct interaction between protein and RNA can be studied using cross-linking immunoprecipitation (CLIP) techniques in living cells. Here, we present a protocol to characterize the direct binding of proteins to RNA:DNA hybrids or RNA-DNA chimeras in living cells using CLIP.

Genotoxic stress impacts pre-mRNA 3'-end processing.

Genotoxic stress, arising from various environmental sources and endogenous cellular processes, pose a constant threat to genomic stability. Cells have evolved intricate mechanisms to detect and repair DNA damage, orchestrating a robust genotoxic stress response to safeguard the integrity of the genome. Recent research has shed light on the crucial role of co- and post-transcriptional regulatory mechanisms in modulating the cellular response to genotoxic stress.

Post-transcriptional gene regulation: From mechanisms to RNA chemistry and therapeutics.

A better understanding of the RNA biology and chemistry is necessary to then develop new RNA therapeutic strategies. This review is the synthesis of a series of conferences that took place during the 6th international course on post-transcriptional gene regulation at Institut Curie. This year, the course made a special focus on RNA chemistry.

Metabolism-dependent secondary effect of anti-MAPK cancer therapy on DNA repair.

Amino acid bioavailability impacts mRNA translation in a codon-dependent manner. Here, we report that the anti-cancer MAPK inhibitors (MAPKi) decrease the intracellular concentration of aspartate and glutamate in melanoma cells. This coincides with the accumulation of ribosomes on codons corresponding to these amino acids and triggers the translation-dependent degradation of mRNAs encoding aspartate- and glutamate-rich proteins, involved in DNA metabolism such as DNA replication and repair.

53BP1 interacts with the RNA primer from Okazaki fragments to support their processing during unperturbed DNA replication.

RNA-binding proteins (RBPs) are found at replication forks, but their direct interaction with DNA-embedded RNA species remains unexplored. Here, we report that p53-binding protein 1 (53BP1), involved in the DNA damage and replication stress response, is an RBP that directly interacts with Okazaki fragments in the absence of external stress. The recruitment of 53BP1 to nascent DNA shows susceptibility to in situ ribonuclease A treatment and is dependent on PRIM1, which synthesizes the RNA primer of Okazaki fragments.

Post-transcriptional checkpoints in autoimmunity.

Post-transcriptional regulation is a fundamental process in gene expression that has a role in diverse cellular processes, including immune responses. A core concept underlying post-transcriptional regulation is that protein abundance is not solely determined by transcript abundance. Indeed, transcription and translation are not directly coupled, and intervening steps occur between these processes, including the regulation of mRNA stability, localization and alternative splicing, which can impact protein abundance.

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.

The multiple roles of LDH in cancer.

High serum lactate dehydrogenase (LDH) levels are typically associated with a poor prognosis in many cancer types. Even the most effective drugs, which have radically improved outcomes in patients with melanoma over the past decade, provide only marginal benefit to those with high serum LDH levels. When viewed separately from the oncological, biochemical, biological and immunological perspectives, serum LDH is often interpreted in very different ways.

Compartment-specific and ELAVL1-coordinated regulation of intronic polyadenylation isoforms by doxorubicin.

Intronic polyadenylation (IPA) isoforms, which contain alternative last exons, are widely regulated in various biological processes and by many factors. However, little is known about their cytoplasmic regulation and translational status. In this study, we provide the first evidence that the genome-wide patterns of IPA isoform regulation during a biological process can be very distinct between the transcriptome and translatome, and between the nucleus and cytosol.

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.

Glycolysis Dependency as a Hallmark of -Mutated Cells.

mutations are recurrent in cancer and result in aberrant splicing of a previously defined set of genes. Here, we investigated the fate of aberrant transcripts induced by mutant SF3B1 and the related functional consequences. We first demonstrate that mutant SF3B1 does not alter global nascent protein synthesis, suggesting target-dependent consequences.

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.

A PD-1/PD-L1 Proximity Assay as a Theranostic Marker for PD-1 Blockade in Patients with Metastatic Melanoma.

Purpose: Less than 50% of patients with melanoma respond to anti-programmed cell death protein 1 (anti-PD-1), and this treatment can induce severe toxicity. Predictive markers are thus needed to improve the benefit/risk ratio of immune checkpoint inhibitors (ICI). Baseline tumor parameters such as programmed death ligand 1 (PD-L1) expression, CD8 T-cell infiltration, mutational burden, and various transcriptomic signatures are associated with response to ICI, but their predictive values are not sufficient.

The plasticity of mRNA translation during cancer progression and therapy resistance.

Translational control of mRNAs during gene expression allows cells to promptly and dynamically adapt to a variety of stimuli, including in neoplasia in response to aberrant oncogenic signalling (for example, PI3K-AKT-mTOR, RAS-MAPK and MYC) and microenvironmental stress such as low oxygen and nutrient supply. Such translational rewiring allows rapid, specific changes in the cell proteome that shape specific cancer phenotypes to promote cancer onset, progression and resistance to anticancer therapies. In this Review, we illustrate the plasticity of mRNA translation.

Non-homologous end-joining at challenged replication forks: an RNA connection?

Defective DNA replication, known as 'replication stress', is a source of DNA damage, a hallmark of numerous human diseases, including cancer, developmental defect, neurological disorders, and premature aging. Recent work indicates that non-homologous end-joining (NHEJ) is unexpectedly active during DNA replication to repair replication-born DNA lesions and to safeguard replication fork integrity. However, erroneous NHEJ events are deleterious to genome stability.

detection of the eIF4F translation initiation complex in mammalian cells and tissues.

The eukaryotic translation initiation complex eIF4F plays an important role in gene expression. The methods that are used to monitor the formation of the eIF4F complex are usually indirect and provide no information on its subcellular localization. This protocol describes a proximity ligation assay-based procedure allowing the direct visualization of the eIF4F complex, as well as its absolute quantification per cell using adapted image analysis software.

Reciprocal Links between Pre-messenger RNA 3'-End Processing and Genome Stability.

The 3'-end processing of most pre-messenger RNAs (pre-mRNAs) involves RNA cleavage and polyadenylation and is coupled to transcription termination. In both yeast and human cells, pre-mRNA 3'-end cleavage is globally inhibited by DNA damage. Recently, further links between pre-mRNA 3'-end processing and the control of genome stability have been uncovered, as reviewed here.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood.

Persistent Cancer Cells: The Deadly Survivors.

Persistent cancer cells are the discrete and usually undetected cells that survive cancer drug treatment and constitute a major cause of treatment failure. These cells are characterized by their slow proliferation, highly flexible energy consumption, adaptation to their microenvironment, and phenotypic plasticity. Mechanisms that underlie their persistence offer highly coveted and sought-after therapeutic targets, and include diverse epigenetic, transcriptional, and translational regulatory processes, as well as complex cell-cell interactions.

Flavaglines as natural products targeting eIF4A and prohibitins: From traditional Chinese medicine to antiviral activity against coronaviruses.

Flavaglines are cyclopenta[b]benzofurans found in plants of the genus Aglaia, several species of which are used in traditional Chinese medicine. These compounds target the initiation factor of translation eIF4A and the scaffold proteins prohibitins-1 and 2 (PHB1/2) to exert various pharmacological activities, including antiviral effects against several types of viruses, including coronaviruses. This review is focused on the antiviral effects of flavaglines and their therapeutic potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).