SRSF6 balances mitochondrial-driven innate immune outcomes through alternative splicing of BAX
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Evaluation Summary:
This paper is of interest to people studying how differentially spliced genes regulate biological processes, and in particular, those interested in the intersection of cell death and immunity. This work offers new insight into how an alternatively spliced protein with a well-known function in cell death regulates the basal expression of genes involved in immunity and sensitizes cells to apoptotic cell death. Overall, the major conclusions are supported by the data but more investigation is needed to support the mechanism by which BAX splicing is inducing the phenotypes observed.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
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.
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Evaluation Summary:
This paper is of interest to people studying how differentially spliced genes regulate biological processes, and in particular, those interested in the intersection of cell death and immunity. This work offers new insight into how an alternatively spliced protein with a well-known function in cell death regulates the basal expression of genes involved in immunity and sensitizes cells to apoptotic cell death. Overall, the major conclusions are supported by the data but more investigation is needed to support the mechanism by which BAX splicing is inducing the phenotypes observed.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
SRSF6 is an understudied SR family member, best characterized for its role in controlling alternative splicing. Through comparative RNA-Seq analysis, the authors find that knockdown of SRSF6 results in a markedly different gene expression program than other SR proteins tested in that SRSF6 depletion leads to a dramatic increase in expression of interferon responsive genes (ISGs) and a downregulation of mitochondrial related genes. Given this correlation the authors explore the possibility that loss of SRSF6 leads to mitochondrial damage, which releases dsDNA to trigger the innate immune response through the DNA-sensor cGAS. They further propose that mitochondrial damage is due to a change in splicing of the gene BAX. The data shown in the manuscript are consistent with these conclusions, however do not rule …
Reviewer #1 (Public Review):
SRSF6 is an understudied SR family member, best characterized for its role in controlling alternative splicing. Through comparative RNA-Seq analysis, the authors find that knockdown of SRSF6 results in a markedly different gene expression program than other SR proteins tested in that SRSF6 depletion leads to a dramatic increase in expression of interferon responsive genes (ISGs) and a downregulation of mitochondrial related genes. Given this correlation the authors explore the possibility that loss of SRSF6 leads to mitochondrial damage, which releases dsDNA to trigger the innate immune response through the DNA-sensor cGAS. They further propose that mitochondrial damage is due to a change in splicing of the gene BAX. The data shown in the manuscript are consistent with these conclusions, however do not rule out additional mechanisms. In particular, the mitochondrial and BAX phenotypes are much less dramatic than the interferon response. Moreover, the authors do not show that the change in BAX splicing induced by loss of SRSF6 is sufficient to lead to a change in ISG expression.
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Reviewer #2 (Public Review):
Wagner, et al, provide strong evidence for Srsf6 as a guard for mitochondrial integrity in macrophages through control of alternative splicing. Prior work identified Srsf6 as important for mitochondrial function in endothelial cells, or in a mouse model of fatty liver disease. This study demonstrates that Srsf6-depleted macrophages exhibit compromised mitochondrial integrity, releasing mitochondrial DNA into the cytosol which triggers innate immune signaling. Consequently, Srsf6-depleted cells produced high levels of Type I interferon in a cGAS-dependent manner. Moreover, a sub-population of Srsf6 KD cells were more susceptible to cell death. The authors test the role of an alternative splice form of Bax, Bax-kappa, as the critical mechanism that leads to increased Type I IFN and cell death in Srsf6-depleted …
Reviewer #2 (Public Review):
Wagner, et al, provide strong evidence for Srsf6 as a guard for mitochondrial integrity in macrophages through control of alternative splicing. Prior work identified Srsf6 as important for mitochondrial function in endothelial cells, or in a mouse model of fatty liver disease. This study demonstrates that Srsf6-depleted macrophages exhibit compromised mitochondrial integrity, releasing mitochondrial DNA into the cytosol which triggers innate immune signaling. Consequently, Srsf6-depleted cells produced high levels of Type I interferon in a cGAS-dependent manner. Moreover, a sub-population of Srsf6 KD cells were more susceptible to cell death. The authors test the role of an alternative splice form of Bax, Bax-kappa, as the critical mechanism that leads to increased Type I IFN and cell death in Srsf6-depleted macrophages.
The many strengths of this well-written and thorough study include rigorous experimental design and hypothesis testing using different methods that employ both gain- and loss- of function. Additionally, the authors use Mycobacterium tuberculosis infection as a physiologically relevant test of their hypothesized role for Srsf6, and find that intracellular bacterial replication and cell death of infected macrophages are significantly impacted. Their proposed model placing Bax-kappa as the central "executioner" when Srsf6 levels or activity are decreased is attractive and supported by much of their data. Some conclusions were less well justified and could be strengthened. Specifically, heterologous expression of Bax-kappa in wildtype cells phenocopied many of the phenotypes of Srsf6-depleted cells, with one notable exception, which is that Bax-kappa mediated cell death in wildtype cells was caspase-dependent, while cell death in Srsf6-depleted cells was Bax-dependent but caspase-independent. While the authors hypothesize this difference is due to Bax-kappa overexpression, this unresolved point weakens the case for the Bax-kappa isoform as the key mechanism for Srsf6KD cell death. Moreover, there were several instances where Srsf6 KD phenotypes appeared statistically significant in one panel but not in another, even using the same assay. Resolving these discrepancies would strengthen the study. Overall, this work reveals an impactful role for SRSF6 in setting thresholds for mitochondrial stress and DAMP release, which can trigger induction of the Type I interferon response during infection.
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Reviewer #3 (Public Review):
The authors identified a splicing factor that regulates mitochondrial homeostasis by regulating the alternative splicing of the pro-apoptotic protein BAX, which induces basal upregulation of interferon stimulated genes and sensitizes cells to apoptotic cell death. They report that loss of Serine/Arginine Rich Splicing factor 6 (SRSF6) results in accumulation of an alternatively spliced form of BAX known as BAX-, which results in increased release of mitochondrial DNA (mtDNA). The released mtDNA is sensed by cGAS, which leads to upregulation of interferon stimulated genes via IRF3. Importantly, the increase in BAX- sensitizes macrophages to apoptosis and various pathogens decreased the expression of SRSF6 during infection, which served a protective role. Interestingly, Mycobacterium tuberculosis decreases …
Reviewer #3 (Public Review):
The authors identified a splicing factor that regulates mitochondrial homeostasis by regulating the alternative splicing of the pro-apoptotic protein BAX, which induces basal upregulation of interferon stimulated genes and sensitizes cells to apoptotic cell death. They report that loss of Serine/Arginine Rich Splicing factor 6 (SRSF6) results in accumulation of an alternatively spliced form of BAX known as BAX-, which results in increased release of mitochondrial DNA (mtDNA). The released mtDNA is sensed by cGAS, which leads to upregulation of interferon stimulated genes via IRF3. Importantly, the increase in BAX- sensitizes macrophages to apoptosis and various pathogens decreased the expression of SRSF6 during infection, which served a protective role. Interestingly, Mycobacterium tuberculosis decreases SRSF6 expression, but this resulted in a replication advantage. Overall, these findings add new mechanistic insight into the role of alternative splicing in regulating immunity and cell death. This work can potentially open novel avenues of inquiry into the role of BAX in regulating apoptosis.
Strengths:
The paper is well written, and the major conclusions are rigorously tested by numerous experiments. The data supports the major conclusions, which are that loss of SRSF6 increases ISG and leads to accumulation of alternatively spliced BAX, sensitizing cells to death.
Weaknesses:
The authors make a very interesting discovery that SRSF6 KD sensitizes macrophages to a caspase independent death by up regulating an alternatively spliced variant of BAX, a protein that has a well-established role in mediating caspase dependent death, but they did not rigorously test whether it was truly caspase independent.
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