Serine/arginine-rich splicing factor 7 promotes the type I interferon response by activating Irf7 transcription.

Tight regulation of macrophage immune gene expression is required to fight infection without risking harmful inflammation. The contribution of RNA-binding proteins (RBPs) to shaping the macrophage response to pathogens remains poorly understood. Transcriptomic analysis reveals that a member of the serine/arginine-rich (SR) family of mRNA processing factors, SRSF7, is required for optimal expression of a cohort of interferon-stimulated genes in macrophages. Using genetic and biochemical assays, we discover that in addition to its canonical role in regulating alternative splicing, SRSF7 drives transcription of interferon regulatory transcription factor 7 (IRF7) to promote antiviral immunity. At the Irf7 promoter, SRSF7 maximizes STAT1 transcription factor binding and RNA polymerase II elongation via cooperation with the H4K20me1 histone methyltransferase KMT5a (SET8). These studies define a role for an SR protein in activating transcription and reveal an RBP-chromatin network that orchestrates macrophage antiviral gene expression.

Serine arginine-rich splicing factor (SRSF7) cooperates with the histone methyltransferase KMT5a to promote the type I interferon response via transcriptional activation of IRF7.

Tight regulation of macrophage immune gene expression is required to fight infection without risking harmful inflammation. The contribution of RNA binding proteins (RBPs) to shaping the macrophage response to pathogens remains poorly understood. Transcriptomic analysis revealed that a member of the serine/arginine-rich (SR) family of mRNA processing factors, SRSF7, is required for optimal expression of a cohort of interferon stimulated genes (ISGs) in macrophages. Using genetic and biochemical assays, we discovered that in addition to its canonical role in regulating alternative splicing, SRSF7 drives transcription of interferon regulatory transcription factor 7 (IRF7) to promote antiviral immunity. At the Irf7 promoter, SRSF7 maximizes STAT1 transcription factor binding and RNA polymerase II elongation via cooperation with the H4K20me1 histone methyltransferase KMT5a (SET8). These studies define an unorthodox role for an SR protein in activating transcription and reveal an unappreciated RNA binding protein-chromatin network that orchestrates macrophage antiviral gene expression.

SRSF6 balances mitochondrial-driven innate immune outcomes through alternative splicing of BAX.

To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage's response to pathogens.

Mitochondrial ROS promotes susceptibility to infection via gasdermin D-mediated necroptosis.

Although mutations in mitochondrial-associated genes are linked to inflammation and susceptibility to infection, their mechanistic contributions to immune outcomes remain ill-defined. We discovered that the disease-associated gain-of-function allele Lrrk2G2019S (leucine-rich repeat kinase 2) perturbs mitochondrial homeostasis and reprograms cell death pathways in macrophages. When the inflammasome is activated in Lrrk2G2019S macrophages, elevated mitochondrial ROS (mtROS) directs association of the pore-forming protein gasdermin D (GSDMD) to mitochondrial membranes. Mitochondrial GSDMD pore formation then releases mtROS, promoting a switch to RIPK1/RIPK3/MLKL-dependent necroptosis. Consistent with enhanced necroptosis, infection of Lrrk2G2019S mice with Mycobacterium tuberculosis elicits hyperinflammation and severe immunopathology. Our findings suggest a pivotal role for GSDMD as an executer of multiple cell death pathways and demonstrate that mitochondrial dysfunction can direct immune outcomes via cell death modality switching. This work provides insights into how LRRK2 mutations manifest or exacerbate human diseases and identifies GSDMD-dependent necroptosis as a potential target to limit Lrrk2G2019S-mediated immunopathology.

Global Transcriptomics Uncovers Distinct Contributions From Splicing Regulatory Proteins to the Macrophage Innate Immune Response.

Pathogen sensing via pattern recognition receptors triggers massive reprogramming of macrophage gene expression. While the signaling cascades and transcription factors that activate these responses are well-known, the role of post-transcriptional RNA processing in modulating innate immune gene expression remains understudied. Given their crucial role in regulating pre-mRNA splicing and other RNA processing steps, we hypothesized that members of the SR/hnRNP protein families regulate innate immune gene expression in distinct ways. We analyzed steady state gene expression and alternatively spliced isoform production in ten SR/hnRNP knockdown RAW 264.7 macrophage-like cell lines following infection with the bacterial pathogen Salmonella enterica serovar Typhimurium (Salmonella). We identified thousands of transcripts whose abundance is increased or decreased by SR/hnRNP knockdown in macrophages. Notably, we observed that SR and hnRNP proteins influence expression of different genes in uninfected versus Salmonella-infected macrophages, suggesting functionalization of these proteins upon pathogen sensing. Likewise, we found that knockdown of SR/hnRNPs promoted differential isoform usage (DIU) for thousands of macrophage transcripts and that these alternative splicing changes were distinct in uninfected and Salmonella-infected macrophages. Finally, having observed a surprising degree of similarity between the differentially expressed genes (DEGs) and DIUs in hnRNP K and U knockdown macrophages, we found that hnRNP K and U knockdown macrophages are both more restrictive to Vesicular Stomatitis Virus (VSV), while hnRNP K knockdown macrophages are more permissive to Salmonella Typhimurium. Based on these findings, we conclude that many innate immune genes evolved to rely on one or more SR/hnRNPs to ensure the proper magnitude of their induction, supporting a model wherein pre-mRNA splicing is critical for regulating innate immune gene expression and controlling infection outcomes in macrophages ex vivo.

The Splicing Factor hnRNP M Is a Critical Regulator of Innate Immune Gene Expression in Macrophages.

While transcriptional control of innate immune gene expression is well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M is a critical repressor of innate immune gene expression and that its function is regulated by pathogen sensing cascades. Loss of hnRNP M led to hyperinduction of a unique regulon of inflammatory and antimicrobial genes following diverse innate immune stimuli. While mutating specific serines on hnRNP M had little effect on its ability to control pre-mRNA splicing or transcript levels of housekeeping genes in resting macrophages, it greatly impacted the protein's ability to dampen induction of specific innate immune transcripts following pathogen sensing. These data reveal a previously unappreciated role for pattern recognition receptor signaling in controlling splicing factor phosphorylation and establish pre-mRNA splicing as a critical regulatory node in defining innate immune outcomes.

Metabolic Rate during a Cognitive Vigilance Challenge at Alternative Workstations.

OBJECTIVE: The aim of this study was to compare energy expenditure (EE, kcal/min) at three workstations during an attention-demanding cognitive function task (Test of Variables of Attention or TOVA). Workstations included the seated desk (SIT), standing desk (STAND), and seated workstation designed to promote spontaneous movement (SWING). METHODS: Adult males (n = 11) and females (n = 13) were assessed for EE using VO2 and VCO2 per quarter of the 22-min TOVA. RESULTS: Average EE were 1.39 ±â€Š0.06 (SIT), 1.55 ±â€Š0.08 (SWING), and 1.44 ±â€Š0.08 (STAND). Main effects (P < 0.05) were seen for workstation (SWING, STAND > SIT), and quarter of TOVA (Q2 < Q1,Q3,Q4). TOVA errors and response times were not different for workstations but increased for Q3 and Q4. CONCLUSION: Spontaneous movement at an alternative workstation elevated EE 10% to 11% compared with sitting and could increase daily nonexercise activity thermogenesis without diminishing mental attention to desk work.

Effect of a novel workstation device on promoting non-exercise activity thermogenesis (NEAT).

BACKGROUND: Strategies to increase non-exercise activity thermogenesis (NEAT) through promotion of movement and energy expenditure at desk stations are needed to help overcome ill effects of prolonged sitting. OBJECTIVE: Examine the metabolic rate during three stages of a workstation: sitting, standing, and use of a device (HOVR®) that promotes leg movement while seated. METHODS: Participants (n = 16; mean ±standard deviation: age 26.1±6.0 years; BMI 24.7±4.3 kg/m2) were tested for VO2 and VCO2 for 15 min at each stage in this order: sitting only, sitting using the HOVR, and standing. Participants performed the same desk work to keep fine-motor activity consistent for the stages. Data collected during the final 5 min of a stage were averaged and analyzed as steady-state data. To evaluate the effect of each stage on cognitive function, the Stroop word-color test was administered after metabolic assessment as the stage continued. One-way ANOVA with repeated measures was used to compare stages for VO2 (L/min), metabolic equivalents (METs), respiratory exchange ratio (RER), and heart rate (p < 0.05). RESULTS: The ANOVA revealed significant differences between the mean values for each stage for each dependent variable (p < 0.05). Post hoc tests indicated VO2 differed for each stage (mean±SD in mL/kg/min: sitting, 4.13±0.56; sitting with HOVR, 4.82±0.74; standing, 4.50±0.53; p < 0.05). METs followed a similar pattern (sitting, 1.19±0.16; sitting with HOVR, 1.39±0.20; standing, 1.29±0.16; p < 0.05). An increase in Stroop Test scores was found as the stages progressed (p < 0.05). CONCLUSION: Modest movement while seated, i.e., use of HOVR, elevated metabolic rate by 17.6% compared to sitting and by 7% compared to standing and might be a reasonable strategy to help elevate NEAT during the workday.