mRNA Splicing-Mediated Gene Expression Regulation in Innate Immunity

At the heart of an inflammatory response lies a tightly regulated gene expression program. Perturbations to this finely tuned response can result in unchecked or inappropriately scaled inflammation, shifting the balance from protective to destructive immunity. A variety of post-transcriptional mechanisms play a role in the fine-tuning of an inflammatory gene expression program. One such mechanism involves unproductive RNA splicing, whereby alternative splicing can frameshift the transcript or introduce a premature termination codon (PTC). These effects render the transcript nonfunctional and/or subject it to nonsense-mediated decay. We observed such an event in Irf7, the master regulator of the type I interferon response. We found a single intron was consistently retained at a level much greater than other introns in the Irf7 transcript. In an effort to understand trans-acting factors that regulate this retention, we used RNA-antisense purification followed by mass spectrometry (RAP-MS) to identify the factor BUD13 as a highly enriched protein on Irf7 transcripts. Deficiency in BUD13 was associated with increased retention, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Beyond this intron retention event in Irf7, we identified a variety of other unproductive splicing events in a number of important genes involved with the innate immune response. This unproductive splicing was not restricted to intron retention events. For example, we identified a frequently used alternative splice site in the crucial murine antiviral response gene, oligoadenylate synthetase 1g (Oas1g) that led to both a frameshift and incorporation of a PTC. Genome editing was used to remove the alternative splice site in a macrophage cell line, which led to both increased Oas1g expression and improved viral clearance. We hypothesize these events exist as a means of mitigation for what might otherwise be an inappropriately scaled response. In doing so, they represent a previously underappreciated layer of gene expression regulation in innate immunity.</p

Alternative Splicing Coupled with Transcript Degradation Modulates OAS1g Antiviral Activity

At the heart of an innate immune response lies a tightly regulated gene expression program. This precise regulation is crucial because small changes can shift the balance from protective to destructive immunity. Here we identify a frequently used alternative splice site in the gene oligoadenylate synthetase 1g (Oas1g), a key component of the 2-5A antiviral system. Usage of this splice site leads to the generation of a transcript subject to decay, and removal of the site leads to increased expression of Oas1g and an improved antiviral response. However, removal of the splice site also leads to an increase in apoptotic cell death, suggesting this splicing event exists as a compromise between the pathogen protective benefits and collateral damage associated with OAS1g activity. Across the innate immune response, we show a multitude of alternative splicing events predicted to lead to decay exist and thus, have the potential to play a significant role in the regulation of gene expression in innate immunity

Bud13 Promotes a Type I Interferon Response By Countering Intron Retention in Irf7

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs

Bud13 Promotes a Type I Interferon Response By Countering Intron Retention in Irf7

Intron retention (IR) has emerged as an important mechanism of gene expression control, but the factors controlling IR events remain poorly understood. We observed consistent IR in one intron of the Irf7 gene and identified BUD13 as an RNA-binding protein that acts at this intron to increase the amount of successful splicing. Deficiency in BUD13 was associated with increased IR, decreased mature Irf7 transcript and protein levels, and consequently a dampened type I interferon response, which compromised the ability of BUD13-deficient macrophages to withstand vesicular stomatitis virus (VSV) infection. Global analysis of BUD13 knockdown and BUD13 cross-linking to RNA revealed a subset of introns that share many characteristics with the one found in Irf7 and are spliced in a BUD13-dependent manner. Deficiency of BUD13 led to decreased mature transcript from genes containing such introns. Thus, by acting as an antagonist to IR, BUD13 facilitates the expression of genes at which IR occurs

Capicua regulates neural stem cell proliferation and lineage specification through control of Ets factors

Capicua (Cic) is a transcriptional repressor mutated in the brain cancer oligodendroglioma. Despite its cancer link, little is known of Cic’s function in the brain. We show that nuclear Cic expression is strongest in astrocytes and neurons but weaker in stem cells and oligodendroglial lineage cells. Using a new conditional Cic knockout mouse, we demonstrate that forebrain-specific Cic deletion increases proliferation and self-renewal of neural stem cells. Furthermore, Cic loss biases neural stem cells toward glial lineage selection, expanding the pool of oligodendrocyte precursor cells (OPCs). These proliferation and lineage effects are dependent on de-repression of Ets transcription factors. In patient-derived oligodendroglioma cells, CIC re-expression or ETV5 blockade decreases lineage bias, proliferation, self-renewal, and tumorigenicity. Our results identify Cic as an important regulator of cell fate in neurodevelopment and oligodendroglioma, and suggest that its loss contributes to oligodendroglioma by promoting proliferation and an OPC-like identity via Ets overactivity

Multi-omic single-cell snapshots reveal multiple independent trajectories to drug tolerance in a melanoma cell line

The determination of individual cell trajectories through a high-dimensional cell-state space is an outstanding challenge for understanding biological changes ranging from cellular differentiation to epigenetic responses of diseased cells upon drugging. We integrate experiments and theory to determine the trajectories that single BRAF^(V600E) mutant melanoma cancer cells take between drug-naive and drug-tolerant states. Although single-cell omics tools can yield snapshots of the cell-state landscape, the determination of individual cell trajectories through that space can be confounded by stochastic cell-state switching. We assayed for a panel of signaling, phenotypic, and metabolic regulators at points across 5 days of drug treatment to uncover a cell-state landscape with two paths connecting drug-naive and drug-tolerant states. The trajectory a given cell takes depends upon the drug-naive level of a lineage-restricted transcription factor. Each trajectory exhibits unique druggable susceptibilities, thus updating the paradigm of adaptive resistance development in an isogenic cell population