Biology subjects

Quesnel-Vallieres, M.

Publications and source records attributed to Quesnel-Vallieres, M..

9 recordsLinked to original sources

bioRxiv2025

Alternate isoforms of IRF7 Differentially Regulate Interferon Expression to Tune Response to Viral Infection

Interferon Regulatory Factor 7 (IRF7), and its homologue IRF3, are master transcriptional regulators of the innate immune response. IRF7 binds to promoters of interferon {beta} (IFN{beta}) and several IFNs as a homodimer or as a heterodimer with IRF3 to drive expression of these type I IFNs, which in turn activate downstream signaling pathways to promote expression of antiviral genes. Here we demonstrate that alternative splicing of the first intron within the coding region of IRF7 is highly regulated across immune tissues and in response to immunologic signals including viral infection. Retention of this intron generates an alternative translation start site, resulting in a N-terminally extended form of the protein (exIRF7) with distinct function from the canonical version of IRF7 (cIRF7). We find that exIRF7 uniquely activates a gene expression program, including IFN{beta}, in response to innate immune triggers. Mechanistically, this enhanced activity of exIRF7 relative to cIRF7 is through increased homodimerization and association with IRF3 on DNA. Furthermore, the enhanced transcriptional activity of exIRF7 controls viral infection to a greater extent than cIRF7, demonstrating that alternative splicing of IRF7 is a previously unrecognized mechanism used by cells to tune the interferon response to control viral infections and other immune challenges. HighlightsO_LIIntron retention in the human IRF7 gene generates a distinct protein isoform that differs in the N-terminus. C_LIO_LIIRF7 intron retention is regulated in a stimuli- and cell-type specific manner. C_LIO_LIThe extended version of IRF7, produced by intron retention, exhibits enhanced transcriptional activation of type I interferon genes. C_LIO_LICells expressing the extended version of IRF7 are more resistant to viral infection. C_LI

molecular biology↗

bioRxiv2025

Neuronal cell adhesion molecule (NRCAM) variant defined by microexon skipping is an essential, antigenically distinct, and targetable surface proteoform in high-grade glioma

To overcome the paucity of known tumor-specific surface antigens in pediatric high-grade glioma (pHGG), we contrasted splicing patterns in pHGGs and normal brain samples. Among alternative splicing events affecting extracellular protein domains, the most pervasive alteration was the skipping of [≤]30 nucleotide-long exons. Several of these skipped microexons mapped to L1-IgCAM family members, such as NRCAM. Bulk and single-nuclei short- and long-read RNA-seq revealed uniform skipping of NRCAM microexons 5 and 19 in virtually every pHGG sample. Importantly, the {Delta}ex5{Delta}ex19 (but not the full-length) NRCAM proteoform was essential for pHGG cell migration and invasion in vitro and tumor growth in vivo. We developed a monoclonal antibody selective for {Delta}ex5{Delta}ex19 NRCAM and demonstrated that "painting" of pHGG cells with this antibody enables killing by T cells armed with an FcRI-based universal immune receptor. Thus, pHGG-specific NRCAM and possibly other L1-IgCAM proteoforms are promising and highly selective targets for adoptive immunotherapies.

cancer biology↗

bioRxiv2023

A Novel Aberrantly Spliced Gain-of-Function NT5C2 Isoform Contributes to Thiopurine Resistance in Acute Lymphoblastic Leukemia

Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B- ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.

cancer biology↗

bioRxiv2023

Targeting mitophagy in SRSF2 mutant hematologic malignancies

Splicing factor mutations are common in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but how they alter cellular functions is unclear. We show that the pathogenic SRSF2P95H/+ mutation disrupts the splicing of mitochondrial mRNAs, impairs mitochondrial complex I function, and robustly increases mitophagy. We also identified a mitochondrial surveillance mechanism by which mitochondrial dysfunction modifies splicing of the mitophagy activator PINK1 to remove a poison intron, increasing the stability and abundance of PINK1 mRNA and protein. SRSF2P95H-induced mitochondrial dysfunction increased PINK1 expression through this mechanism, which is essential for survival of SRSF2P95H/+ cells. Inhibition of splicing with a glycogen synthase kinase 3 inhibitor promoted retention of the poison intron, impairing mitophagy and activating apoptosis in SRSF2P95H/+ cells. These data reveal a homeostatic mechanism for sensing mitochondrial stress through PINK1 splicing and identify increased mitophagy as a disease marker and a therapeutic vulnerability in SRSF2P95H mutant MDS and AML.

cancer biology↗

bioRxiv2021

Identifying common transcriptome signatures of cancer by interpreting deep learning models

Cancer is a set of diseases characterized by unchecked cell proliferation and invasion of surrounding tissues. The many genes that have been genetically associated with cancer or shown to directly contribute to oncogenesis vary widely between tumor types, but common gene signatures that relate to core cancer pathways have also been identified, signifying that cancer cases display common hallmark molecular features. It is not clear however whether there exist additional sets of genes or transcriptomic features that are less well known in cancer biology but that are also commonly deregulated across several cancer types. Here, in order to agnostically identify transcriptomic features that are commonly shared between cancer types, we used RNA-Seq datasets encompassing thousands of samples from 19 healthy tissue types and 18 solid tumor types to train three feed-forward neural networks, based either on protein-coding gene expression, lncRNA expression or splice junction use, to distinguish between healthy and tumor samples. All three models achieve high precision, recall and accuracy on test sets derived from 13 datasets used during training and on an independent test dataset, indicating that our models recognize transcriptome signatures that are consistent across tumors. Analysis of attribution values extracted from our models reveals that genes that are commonly altered in cancer by expression or splicing variations are under strong evolutionary and selective constraints, suggesting that they have important cellular functions. Importantly, we found that genes composing our cancer transcriptome signatures are not frequently affected by mutations or genomic alterations and that their functions differ widely from the genes genetically associated with cancer. Finally, our results also highlighted that deregulation of RNA-processing genes and aberrant splicing are pervasive features across a large array of solid tumor types. The transcriptomic features that we highlight here define cancer signatures that may reflect causal variations or consequences of disease state, or a combination of both.

cancer biology↗

bioRxiv2021

High-throughput mutagenesis identifies mutations and RNA binding proteins controlling CD19 splicing and CART-19 therapy resistance

During CART-19 immunotherapy for B-cell acute lymphoblastic leukaemia (B-ALL), many patients relapse due to loss of the cognate CD19 epitope. Since epitope loss can be caused by aberrant CD19 exon 2 processing, we herein investigate the regulatory code that controls CD19 splicing. We combine high-throughput mutagenesis with mathematical modelling to quantitatively disentangle the effects of all mutations in the region comprising CD19 exons 1-3. Thereupon, we identify ~200 single point mutations that alter CD19 splicing and thus could predispose B-ALL patients to CART-19 resistance. Furthermore, we report almost 100 previously unknown splice isoforms that emerge from cryptic splice sites and likely encode non-functional CD19 proteins. We further identify cis-regulatory elements and trans-acting RNA-binding proteins that control CD19 splicing (e.g., PTBP1 and SF3B4) and validate that loss of these factors leads to enhanced CD19 mis-splicing. Our dataset represents a comprehensive resource for potential prognostic factors predicting success of CART-19 therapy. HighlightsO_LIMutations in relapsed CART-19 patients lead to CD19 mis-splicing C_LIO_LIHigh-throughput mutagenesis uncovers ~200 single point mutations with a potential role in CART-19 therapy resistance C_LIO_LIMany mutations generate non-functional CD19 proteins by activating cryptic splice sites C_LIO_LIRNA-binding proteins such as PTBP1 are key to the expression of properly spliced, CART-19 immunotherapy-sensitive isoforms C_LI

molecular biology↗

bioRxiv2021

The Global Protein-RNA Interaction map of Epithelial Splicing Regulatory Protein 1 defines a post-transcriptional program that is essential for epithelial cell function

The epithelial splicing regulatory proteins, ESRP1 and ESRP2 are essential for mammalian development through regulation of a global program of alternative splicing of genes involved in maintenance of epithelial cell function. To further inform our understanding of the molecular functions of ESRP1 we performed enhanced crosslinking immunoprecipitation coupled with high throughput sequencing (eCLIP) in epithelial cells of mouse epidermis. The genome-wide binding sites of ESRP1 were integrated with RNA-Seq analysis of alterations in splicing and total gene expression that result from epidermal ablation of Esrp1 and Esrp2. These studies demonstrated that ESRP1 functions in splicing regulation occur primarily through direct binding in a position-dependent manner to either promote exon inclusion or skipping. In addition, we also identified widespread binding of ESRP1 in 3 and 5 untranslated regions (UTRs) of genes involved in epithelial cell function suggesting that its post-transcriptional functions extend beyond splicing regulation.

molecular biology↗

bioRxiv2020

Alternative splicing redefines landscape of commonly mutated genes in acute myeloid leukemia

Most genes associated with Acute Myeloid Leukemia (AML) are mutated in less than 10% of patients, suggesting alternative mechanisms for gene disruption contribute to this disease. Here we find a set of splicing events that disrupt the expression of a subset of AML-associated genes, including EZH2 and ZRSR2, independent of known somatic mutations. Most strikingly, in at least one cohort, aberrant splicing triples the number of patients with a reduction in functional EZH2 as compared to that predicted by somatic mutation of EZH2 alone. Together, these results demonstrate that classical mutation analysis underestimates the burden of functional gene disruption in AML and highlights the importance of assessing the contribution of alternative splicing to gene dysregulation in human disease.

genomics↗

bioRxiv2020

Rapid and scalable profiling of nascent RNA with fastGRO

Genome-wide profiling of nascent RNA has become a fundamental tool to study transcription regulation. Over the past decade, next-generation sequencing has fostered development of a handful of techniques (i.e. GRO-seq, PRO-seq, TT-seq and NET-seq) that map unprocessed transcripts originating from both the coding and the noncoding portion of the genome. Unlike steady-state RNA sequencing, nascent RNA profiling mirrors the real-time activity of RNA Polymerases and provides an accurate readout of transcriptome-wide variations that occur during short time frames (i.e. response to external stimuli or rapid metabolic changes). Some species of nuclear RNAs, albeit functional, have a short half-life and can only be accurately gauged by nascent RNA techniques (i.e. lincRNAs and eRNAs). Furthermore, these techniques capture uncapped post-cleavage RNA at termination sites or promoter-associated antisense RNAs, providing a unique insight into RNAPII dynamics and processivity. Here we present a run-on assay with 4s-UTP labelling, followed by reversible biotinylation and affinity purification via streptavidin. Our protocol allows streamlined sample preparation within less than 3 days. We named the technique fastGRO (fast Global Run-On). We show that fastGRO is highly reproducible and yields a more complete and extensive coverage of nascent RNA than comparable techniques. Importantly, we demonstrate that fastGRO is scalable and can be performed with as few as 0.5x10^6 cells.

genomics↗