Fatty Acid Synthase associates with nuclear-derived cytoplasmic dsRNA molecules and influences antiviral innate immune response

Article activity feed

1. 

   Review Commons

   Jul 31, 2026

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Reviewers’ comments:

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   This manuscript identifies a potential connection between FASN, endogenous dsRNAs, and innate immune signaling. The authors show that FASN depletion increases cytoplasmic dsRNA staining, enhances IFN transcription and restricts Sindbis virus replication. Overall, the basic observation seems to be solid and well-controlled. Depending on which journal this is being considered for, the central mechanism could be better developed and elucidated to entail more molecular details. As it currently stands, several important questions, especially ones regarding the FASN-RNA …

   Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Reply to the reviewers

   Reviewers’ comments:

   Reviewer #1 (Evidence, reproducibility and clarity (Required)):

   This manuscript identifies a potential connection between FASN, endogenous dsRNAs, and innate immune signaling. The authors show that FASN depletion increases cytoplasmic dsRNA staining, enhances IFN transcription and restricts Sindbis virus replication. Overall, the basic observation seems to be solid and well-controlled. Depending on which journal this is being considered for, the central mechanism could be better developed and elucidated to entail more molecular details. As it currently stands, several important questions, especially ones regarding the FASN-RNA interactions remain unresolved.

   Major comments

   The conclusion that FASN regulates dsRNA accessibility rather than dsRNA abundance requires further support. The authors show increased J2 staining without major changes in the global dsRNA landscape by J2-RIP-seq. However, alternative explanations could be: (1) changes in dsRNA structure or length distribution could affect J2 recognition, i.e. epitope masked by FASN binding; (2) differences in RNA-protein complex assembly could influence J2 accessibility; (3) Altered subcellular localization of dsRNAs could increase immunofluorescence signals without substantially affecting RIP-seq recovery.

   1. The nature of the FASN-RNA interaction remains unclear. The authors demonstrate that FASN associates with a subset of endogenous RNAs, but it is not clear whether this interaction is sequence-specific, structure-specific, or largely nonspecific. Are there common sequence motifs, secondary structures, repetitive elements, or specific transcript classes enriched among FASN-associated RNAs? Without a clearer understanding of RNA selectivity, it remains difficult to evaluate the biological significance of the observed interactions.
   2. The study would be significantly strengthened by complementation experiments. Although the FASN knockout phenotype is convincing, add-back experiments are needed to demonstrate specificity and probe potential mechanisms. Reconstitution with wild-type FASN should rescue the dsRNA and immune phenotypes. Furthermore, domain-specific mutants or catalytic mutants (e.g., palmitoylation) could help distinguish whether the observed effects depend on FASN enzymatic activity, a specific protein domain, or a non-canonical RNA-binding function. Such experiments would greatly improve the mechanistic depth of the study. It will also address whether the observed dsRNA increase phenotype is irreversible.

   The manuscript proposes that increased endogenous dsRNA sensing drives the inflammatory phenotype in FASN-deficient cells. However, direct evidence for enhanced PRR engagement is currently lacking. Although MDA5 and MAVS knockdown experiments suggest a possible contribution of this pathway, the observed knockdown effects are relatively modest and needs to be repeated by CRISPR knockout. Does FASN, MAVS double knockout cells rescue the slow growth phenotype? In addition, direct measurements of PRR activation, such as MDA5-RNA association, MAVS activation, IRF3 phosphorylation, or related downstream signaling events, were not examined.

   1. The antiviral phenotype observed during Sindbis virus infection is interesting, but the underlying mechanism remains uncertain. The authors propose that enhanced endogenous dsRNA sensing contributes to viral restriction, yet alternative explanations related to altered lipid metabolism, impaired membrane remodeling, or other consequences of FASN deficiency cannot be excluded. Especially when MDA5 and MAVS knockdown effect appears modest. Additional experiments disrupting interferon signaling or innate immune sensing pathways using the FASN, MAVS double knockout cells would help determine whether the antiviral phenotype is directly linked to the proposed dsRNA sensing mechanism.

   Reviewer #1 (Significance (Required)):

   This manuscript identifies a potential connection between FASN, endogenous dsRNAs, and innate immune signaling. The authors show that FASN depletion increases cytoplasmic dsRNA staining, enhances IFN transcription and restricts Sindbis virus replication. Overall, the basic observation seems to be solid and well-controlled. Depending on which journal this is being considered for, the central mechanism could be better developed and elucidated to entail more molecular details. As it currently stands, several important questions, especially ones regarding the FASN-RNA interactions remain unresolved.

   Reviewer #2 (Evidence, reproducibility and clarity (Required)):

   The manuscript by Pasquier and colleagues investigates the role of fatty acid synthase (FASN) in the regulation of innate immune signaling through its interaction with endogenous dsRNAs. The authors build upon their previous study, in which they identified FASN as a potential dsRNA-binding protein. They show that FASN knockout results in increased cytoplasmic dsRNA detection accompanied by the induction of inflammation-related genes. Subsequent multi-omics analyses reveal that FASN associates with dsRNAs derived from nuclear genome-encoded transcripts in multiple contexts. Interestingly, FASN depletion does not alter the overall expression of these dsRNAs but increases their detection by the J2 antibody. Functionally, FASN depletion enhances innate immune responses to dsRNA stimulation and Sindbis virus infection.

   While the study provides evidence supporting an proviral role for FASN, the manuscript lacks mechanistic insight into this phenomenon. In particular, it remains unclear how FASN depletion enhances dsRNA detection without altering endogenous dsRNA abundance. The following points should be addressed:

   1. The main limitation of the manuscript is the absence of a mechanistic explanation for how FASN depletion leads to increased dsRNA detection without affecting expression. Does the increased dsRNA detection require a direct interaction between FASN and dsRNAs? The authors should provide additional evidence to clarify whether FASN binding masks dsRNA epitopes or regulates dsRNA accessibility to recognition by J2 and innate immune sensors.

   1. The authors show that FASN directly binds nuclear genome-encoded transcripts. It would be important to perform RNA-FISH for selected FASN-interacting RNAs to determine whether their cellular localization or J2-detectable signal is altered in FASN-depleted cells.

   1. In Figure 2B, the authors demonstrate enrichment of inflammation-related gene signatures in FASN knockout cells. However, the magnitude of induction is not clear. How does the inflammatory response compare with canonical innate immune activation, such as viral infection or poly(I:C) stimulation? Although Figure 2A partially addresses this point, many of the highlighted genes are not directly associated with inflammatory responses.

   2. For the transcripts directly associated with FASN, do they contain predicted dsRNA-forming regions or known structured RNA elements? The current data do not sufficiently demonstrate whether these transcripts indeed form dsRNA structures. Although some are enriched in J2 immunoprecipitation, additional structural analyses, including computational prediction of dsRNA regions, would strengthen the conclusion.

   3. The authors should further investigate whether the enhanced antiviral activity observed upon FASN depletion is mediated by elevated endogenous dsRNA sensing. Are dsRNA sensors such as PKR, MDA5, or RIG-I more strongly activated in FASN knockout cells? Do these sensors exhibit increased binding to endogenous dsRNAs following FASN depletion?

   Reviewer #2 (Significance (Required)):

   Main strength: Uncover the proviral role of FASN via regulation of endogenous dsRNAs

   Limitations: Molecular mechanism on how FASN depletion increases the accessibility of dsRNAs without affecting their expression. Also, need better characterization of FASN-binding RNAs, especially their secondary structure.

   Reviewer #3 (Evidence, reproducibility and clarity (Required)):

   Summary The manuscript entitled, "Fatty Acid Synthase associates with nuclear-derived cytoplasmic dsRNA molecules and influences antiviral innate immune response," by Pasquier et al. identifies an unexpected function for FASN as an endogenous dsRNA-associated protein and explores the relationship between FASN, cellular dsRNA homeostasis, and antiviral innate immunity. Using FASN knockout cells, the authors demonstrate that depletion of FASN results in accumulation of cellular dsRNA, characterize the endogenous dsRNA species associated with FASN, and show that loss of FASN is accompanied by increased expression of interferon-stimulated genes and restriction of SINV replication. The biochemical characterization of FASN-associated dsRNAs together with the accompanying transcriptomic analyses provide compelling evidence that FASN interacts with a distinct subset of endogenous dsRNAs, representing a novel function for this metabolic enzyme.

   While evidence is presented that FASN can associate with endogenous dsRNA, several aspects of the proposed functional model extend beyond what is directly demonstrated by the data. In particular, the manuscript presents dsRNA accumulation, innate immune activation, and viral restriction as a consequence of FASN-mediated dsRNA regulation. However, it remains unclear whether these downstream phenotypes arise specifically from the newly described RNA-binding activity of FASN or instead reflect broader cellular consequences of FASN deficiency, including alterations in lipid metabolism and protein palmitoylation. Likewise, although the authors identify a population of FASN-associated dsRNAs, the relationship between these transcripts and the dsRNAs that accumulate following FASN depletion remains insufficiently resolved, making it difficult to mechanistically understand how FASN regulates dsRNA homeostasis. Overall, the manuscript provides a number of intriguing observations regarding FASN association with endogenous dsRNA. However, the functional relationship between these findings and the proposed effects on dsRNA homeostasis and innate immunity would benefit from additional clarification before the mechanistic model can be fully supported.

   Major Concerns

   It remains unclear whether the inflammatory and antiviral phenotypes observed following FASN depletion are attributable to FASN's newly described dsRNA-binding activity or to the broader metabolic consequences of losing FASN. The manuscript presents dsRNA accumulation, induction of ISGs, and restriction of SINV as downstream consequences of FASN-mediated dsRNA regulation. While these observations are compelling, they remain correlative and do not distinguish between effects arising from dsRNA regulation versus the numerous metabolic changes expected following loss of FASN. In particular, reduced palmitate production and altered protein palmitoylation are well-established consequences of FASN depletion and have documented roles in MAVS signaling and antiviral immunity. This distinction is important because the principal claim of the manuscript is not simply that FASN depletion alters innate immunity, but that its RNA-binding activity is proposed to underlie these effects. At present, the data support an association between these phenotypes but fall somewhat short of demonstrating that the observed immune response is specifically driven by FASN-mediated regulation of endogenous dsRNA.

   1. While the manuscript does provide evidence that FASN associates with a subset of endogenous dsRNAs, the relationship between these RNAs and those that accumulate following FASN depletion remains unclear. The authors conclude that FASN regulates dsRNA homeostasis, yet also show that transcripts associated with FASN do not measurably accumulate in knockout cells. This creates a disconnect between RNA association and functional regulation that is not adequately resolved.

   An overlap analysis comparing the dsRNA populations identified in wt and KO cells would help clarify whether FASN regulates a stable population of transcripts or whether broader remodeling of the dsRNA landscape occurs following FASN loss. More generally, the manuscript would be stronger if it helps clarify or distinguish the observation that FASN binds endogenous dsrna from the conclusion that it regulates cellular dsRNA homeostasis; the latter is not fully supported with the presented data.

   Minor concerns

   The mitochondrial localization of dsRNA needs additional validation. Specifically, Fig 3A requires quantitative analysis of mitochondrial abundance, while Fig 3C could be strengthened through orthogonal validation of mitochondrial localization (e.g., microscopy-based co-localization or further purification of the mitochondrial fraction). Inclusion of an RNase control would also help demonstrate that the enrichment is specific to dsRNA.

   The role of the ADAR depletion experiments within the overall narrative is somewhat unclear. Consider either integrating these data more directly into the proposed model or reducing their emphasis. Along similar lines, Fig 4B should clarify whether the increased FASN-dsRNA association reflects greater binding or simply increased abundance of both FASN and dsRNA following 5-Aza treatment. • Several aspects of the data presentation needs clarification. These include the rationale for selecting IFIT1 for validation (Fig 2A), the relationship between proteins highlighted in the Fig 4 volcano plot and those selected for immunoblot validation, the apparent reduction in tubulin complicating interpretation of the SINV capsid blot (Fig 5B), and the conclusion that J2-positive puncta are distinct from SINV RNA without co-localization analysis. • Several figures could be improved for presentation. Quantification of the J2 signal in Fig S1C (analogous to Fig 1D) and improved labeling of the volcano plots would improve readability. • Several minor editorial revisions are recommended. These include describing ACP as a carrier/tethering rather than catalytic domain, citing Fig S2 in the Results, defining the long form of NES in the Fig 2 legend, removing redundant panel descriptions in the Fig 3G legend, correcting the missing text on page 14, and resolving the duplicated figures and formatting issues present throughout the manuscript.

   Reviewer #3 (Significance (Required)):

   Strengths Nice extension of FASN biology beyond its canonical metabolic role Introduces FASN as a potential endogenous dsRNA-binding protein Broad interest for the RNA biology and innate immunity communities

   Limitations RNA binding not fully demonstrated with higher-resolution approaches such as more precise CLIP approaches

   Mechanistic link between FASN dsRNA homeostasis and innate immune activation remains incomplete

   Advance Good conceptual advance but is more descritive than mechanistic

   Audience: RNA biology RNA-binding proteins Innate immunity Host-virus interactions Moonlighting metabolic enzymes

   Revision plan:

   *1. *General Statements:

   We thank the three reviewers for their careful evaluation of our manuscript and for their constructive comments. We are encouraged that all reviewers considered the central observations of the study to be robust and appreciated the conceptual advance provided by the identification of a previously unrecognized association between FASN and endogenous dsRNAs, together with its impact on antiviral innate immunity.

   We also acknowledge the fact that the main limitation of the current manuscript is the mechanistic understanding of how FASN regulates endogenous dsRNA accessibility and how this relates to innate immune activation. We agree that strengthening the mechanistic aspects of the study will substantially improve the manuscript. Nonetheless, dissecting the full mechanism may require more time which goes beyond the scope of this paper.

   Based on the comments of the three reviewers, our revision plan will therefore focus on three major objectives:

   • further characterizing the endogenous RNAs associated with FASN,
   • strengthening the mechanistic aspects linking increased accessibility of endogenous dsRNAs to FASN depletion,
   • providing additional evidence linking endogenous dsRNA accessibility to innate immune priming and antiviral activity. Importantly, we also intend to clarify our proposed model. Our data do not suggest that loss of FASN induces a strong spontaneous interferon response comparable to that triggered by viral infection or poly(I:C) stimulation. We rather propose that FASN depletion establishes a primed state by increasing the accessibility of a subset of endogenous dsRNAs to innate immune sensors, thereby lowering the threshold for antiviral activation upon subsequent challenge. We will revise the manuscript to better communicate this model, improving data presentation throughout the manuscript and discussion to clearly distinguish experimentally supported conclusions from mechanistic interpretations.

   __2. __Description of the planned revisions:

   Further characterization of FASN-associated endogenous RNAs

   To better define the RNA population associated with FASN, we will perform additional bioinformatic analyses using our existing RIP-seq datasets. These analyses will include:

   • characterization of transcript classes (coding and non-coding RNAs);
   • analysis of sequence composition and motif enrichment;
   • prediction of RNA structural features (including GC content, normalized minimum free energy, paired nucleotide fraction and predicted stem length);
   • analysis of overlap with repetitive elements (including Alu, LINE and LTR elements);
   • comparison of predicted structural and sequence features of FASN-associated RNAs and J2-enriched RNAs;
   • additional comparisons between dsRNA populations identified in wild-type and FASN knockout cells. These analyses will help determine whether FASN preferentially associates with specific classes of endogenous RNAs. Based on the shortlisted RNAs, we will also test selected FASN-associated transcripts by RT-qPCR following RNase III treatment, to determine whether they are forming dsRNA with or without FASN depletion.

   • Strengthening the link between endogenous dsRNA accessibility and innate immune activation To provide more evidence that increased accessibility of endogenous dsRNAs results in enhanced innate immune sensing, we plan to further examine activation of dsRNA sensing pathways.

   Specifically, we will assess activation of downstream signaling components (including IRF3 activation) and further investigate the association of endogenous dsRNAs with innate immune sensors by examining MDA5 recruitment to J2-positive dsRNA complexes. Where technically feasible, we will also evaluate the spatial proximity between endogenous dsRNAs and MDA5 by microscopy-based approaches.

   To strengthen the link between endogenous dsRNA sensing and the primed inflammatory phenotype, we also plan to generate polyclonal CRISPR/Cas9 MDA5 or MAVS knockout cell populations in combination with FASN depletion by siRNA treatment, and evaluate the effects on interferon-responsive gene expression such as IFIT1.

   • Relationship between RNA binding and the metabolic functions of FASN Several reviewers raised the important question of whether the observed immune phenotypes arise from FASN-mediated regulation of endogenous dsRNAs or from broader metabolic consequences of FASN deficiency.

   To address this point, we plan to complement our genetic analyses with pharmacological inhibition of FASN catalytic activity using independent inhibitors and assess their effects on endo-dsRNA accumulation and Sindbis virus replication. We also plan to evaluate whether supplementation with exogenous palmitate rescues the observed phenotypes. These experiments will help distinguish catalytic from non-canonical functions of FASN in regulating endo-dsRNA accessibility and antiviral responses.

   Where feasible, we will also attempt complementation experiments using ectopic expression of wild-type FASN in FASN knockout cells.

   Additional manuscript improvements

   We will incorporate the requested additional quantifications, improve figure presentation and labeling, clarify the rationale for selected validation experiments, expand the discussion of alternative mechanistic models, and address the editorial and presentation issues identified by the reviewers.

   __3. __Description of analyses that authors prefer not to carry out:

   Although we agree that additional mechanistic studies would further strengthen the manuscript, we believe that several of the experiments suggested by the reviewers extend beyond the scope of this work.

   In particular, generation and characterization of multiple FASN domain-specific or RNA-binding mutants is currently not feasible. FASN is a large multifunctional enzyme containing several catalytic domains and no canonical RNA-binding domain has yet been defined. Consequently, designing mutants that specifically disrupt RNA binding while preserving enzymatic activities would be possible but difficult to deliver within the timeframe of this revision. Instead, we propose to address the reviewers' concerns through complementary pharmacological approaches (using FASN inhibitors), palmitate supplementation experiments and wild-type FASN complementation where feasible.

   Similarly, while high-resolution CLIP approaches would provide valuable information regarding FASN binding sites on the RNA, these technically demanding experiments fall beyond the scope of the current revision. We believe that the additional computational analyses, biochemical validation and functional experiments described above will substantially strengthen the conclusions regarding the interaction between FASN and endogenous structured RNAs.

   Finally, although generation of stable double knockout cell lines (e.g. FASN/MAVS or FASN/MDA5) could provide additional mechanistic insight, we consider that this could take too much time. Instead, we plan to generate polyclonal CRISPR/Cas9 knockout populations of MAVS or MDA5 in combination with siFASN to strengthen the causal relationship between endogenous dsRNA sensing and the observed primed immune phenotype.

   Read the original source
2. 

   Review Commons

   Jul 31, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   Summary

   The manuscript entitled, "Fatty Acid Synthase associates with nuclear-derived cytoplasmic dsRNA molecules and influences antiviral innate immune response," by Pasquier et al. identifies an unexpected function for FASN as an endogenous dsRNA-associated protein and explores the relationship between FASN, cellular dsRNA homeostasis, and antiviral innate immunity. Using FASN knockout cells, the authors demonstrate that depletion of FASN results in accumulation of cellular dsRNA, characterize the endogenous dsRNA species associated with FASN, and show that loss of FASN is accompanied by increased expression of …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #3

   Evidence, reproducibility and clarity

   Summary

   The manuscript entitled, "Fatty Acid Synthase associates with nuclear-derived cytoplasmic dsRNA molecules and influences antiviral innate immune response," by Pasquier et al. identifies an unexpected function for FASN as an endogenous dsRNA-associated protein and explores the relationship between FASN, cellular dsRNA homeostasis, and antiviral innate immunity. Using FASN knockout cells, the authors demonstrate that depletion of FASN results in accumulation of cellular dsRNA, characterize the endogenous dsRNA species associated with FASN, and show that loss of FASN is accompanied by increased expression of interferon-stimulated genes and restriction of SINV replication. The biochemical characterization of FASN-associated dsRNAs together with the accompanying transcriptomic analyses provide compelling evidence that FASN interacts with a distinct subset of endogenous dsRNAs, representing a novel function for this metabolic enzyme. While evidence is presented that FASN can associate with endogenous dsRNA, several aspects of the proposed functional model extend beyond what is directly demonstrated by the data. In particular, the manuscript presents dsRNA accumulation, innate immune activation, and viral restriction as a consequence of FASN-mediated dsRNA regulation. However, it remains unclear whether these downstream phenotypes arise specifically from the newly described RNA-binding activity of FASN or instead reflect broader cellular consequences of FASN deficiency, including alterations in lipid metabolism and protein palmitoylation. Likewise, although the authors identify a population of FASN-associated dsRNAs, the relationship between these transcripts and the dsRNAs that accumulate following FASN depletion remains insufficiently resolved, making it difficult to mechanistically understand how FASN regulates dsRNA homeostasis. Overall, the manuscript provides a number of intriguing observations regarding FASN association with endogenous dsRNA. However, the functional relationship between these findings and the proposed effects on dsRNA homeostasis and innate immunity would benefit from additional clarification before the mechanistic model can be fully supported.

   Major Concerns

   1. It remains unclear whether the inflammatory and antiviral phenotypes observed following FASN depletion are attributable to FASN's newly described dsRNA-binding activity or to the broader metabolic consequences of losing FASN. The manuscript presents dsRNA accumulation, induction of ISGs, and restriction of SINV as downstream consequences of FASN-mediated dsRNA regulation. While these observations are compelling, they remain correlative and do not distinguish between effects arising from dsRNA regulation versus the numerous metabolic changes expected following loss of FASN. In particular, reduced palmitate production and altered protein palmitoylation are well-established consequences of FASN depletion and have documented roles in MAVS signaling and antiviral immunity.

   This distinction is important because the principal claim of the manuscript is not simply that FASN depletion alters innate immunity, but that its RNA-binding activity is proposed to underlie these effects. At present, the data support an association between these phenotypes but fall somewhat short of demonstrating that the observed immune response is specifically driven by FASN-mediated regulation of endogenous dsRNA.

   1. While the manuscript does provide evidence that FASN associates with a subset of endogenous dsRNAs, the relationship between these RNAs and those that accumulate following FASN depletion remains unclear. The authors conclude that FASN regulates dsRNA homeostasis, yet also show that transcripts associated with FASN do not measurably accumulate in knockout cells. This creates a disconnect between RNA association and functional regulation that is not adequately resolved.

   An overlap analysis comparing the dsRNA populations identified in wt and KO cells would help clarify whether FASN regulates a stable population of transcripts or whether broader remodeling of the dsRNA landscape occurs following FASN loss. More generally, the manuscript would be stronger if it helps clarify or distinguish the observation that FASN binds endogenous dsrna from the conclusion that it regulates cellular dsRNA homeostasis; the latter is not fully supported with the presented data.

   Minor concerns

   • The mitochondrial localization of dsRNA needs additional validation. Specifically, Fig 3A requires quantitative analysis of mitochondrial abundance, while Fig 3C could be strengthened through orthogonal validation of mitochondrial localization (e.g., microscopy-based co-localization or further purification of the mitochondrial fraction). Inclusion of an RNase control would also help demonstrate that the enrichment is specific to dsRNA.
   • The role of the ADAR depletion experiments within the overall narrative is somewhat unclear. Consider either integrating these data more directly into the proposed model or reducing their emphasis. Along similar lines, Fig 4B should clarify whether the increased FASN-dsRNA association reflects greater binding or simply increased abundance of both FASN and dsRNA following 5-Aza treatment.
   • Several aspects of the data presentation needs clarification. These include the rationale for selecting IFIT1 for validation (Fig 2A), the relationship between proteins highlighted in the Fig 4 volcano plot and those selected for immunoblot validation, the apparent reduction in tubulin complicating interpretation of the SINV capsid blot (Fig 5B), and the conclusion that J2-positive puncta are distinct from SINV RNA without co-localization analysis.
   • Several figures could be improved for presentation. Quantification of the J2 signal in Fig S1C (analogous to Fig 1D) and improved labeling of the volcano plots would improve readability.
   • Several minor editorial revisions are recommended. These include describing ACP as a carrier/tethering rather than catalytic domain, citing Fig S2 in the Results, defining the long form of NES in the Fig 2 legend, removing redundant panel descriptions in the Fig 3G legend, correcting the missing text on page 14, and resolving the duplicated figures and formatting issues present throughout the manuscript.

   Significance

   Strengths

   Nice extension of FASN biology beyond its canonical metabolic role Introduces FASN as a potential endogenous dsRNA-binding protein Broad interest for the RNA biology and innate immunity communities

   Limitations

   RNA binding not fully demonstrated with higher-resolution approaches such as more precise CLIP approaches Mechanistic link between FASN dsRNA homeostasis and innate immune activation remains incomplete

   Advance

   Good conceptual advance but is more descritive than mechanistic

   Audience

   RNA biology RNA-binding proteins Innate immunity Host-virus interactions Moonlighting metabolic enzymes

   Read the original source
3. 

   Review Commons

   Jul 31, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The manuscript by Pasquier and colleagues investigates the role of fatty acid synthase (FASN) in the regulation of innate immune signaling through its interaction with endogenous dsRNAs. The authors build upon their previous study, in which they identified FASN as a potential dsRNA-binding protein. They show that FASN knockout results in increased cytoplasmic dsRNA detection accompanied by the induction of inflammation-related genes. Subsequent multi-omics analyses reveal that FASN associates with dsRNAs derived from nuclear genome-encoded transcripts in multiple contexts. Interestingly, FASN depletion does not alter the overall …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #2

   Evidence, reproducibility and clarity

   The manuscript by Pasquier and colleagues investigates the role of fatty acid synthase (FASN) in the regulation of innate immune signaling through its interaction with endogenous dsRNAs. The authors build upon their previous study, in which they identified FASN as a potential dsRNA-binding protein. They show that FASN knockout results in increased cytoplasmic dsRNA detection accompanied by the induction of inflammation-related genes. Subsequent multi-omics analyses reveal that FASN associates with dsRNAs derived from nuclear genome-encoded transcripts in multiple contexts. Interestingly, FASN depletion does not alter the overall expression of these dsRNAs but increases their detection by the J2 antibody. Functionally, FASN depletion enhances innate immune responses to dsRNA stimulation and Sindbis virus infection.

   While the study provides evidence supporting an proviral role for FASN, the manuscript lacks mechanistic insight into this phenomenon. In particular, it remains unclear how FASN depletion enhances dsRNA detection without altering endogenous dsRNA abundance. The following points should be addressed:

   1. The main limitation of the manuscript is the absence of a mechanistic explanation for how FASN depletion leads to increased dsRNA detection without affecting expression. Does the increased dsRNA detection require a direct interaction between FASN and dsRNAs? The authors should provide additional evidence to clarify whether FASN binding masks dsRNA epitopes or regulates dsRNA accessibility to recognition by J2 and innate immune sensors.

   2. The authors show that FASN directly binds nuclear genome-encoded transcripts. It would be important to perform RNA-FISH for selected FASN-interacting RNAs to determine whether their cellular localization or J2-detectable signal is altered in FASN-depleted cells.

   3. In Figure 2B, the authors demonstrate enrichment of inflammation-related gene signatures in FASN knockout cells. However, the magnitude of induction is not clear. How does the inflammatory response compare with canonical innate immune activation, such as viral infection or poly(I:C) stimulation? Although Figure 2A partially addresses this point, many of the highlighted genes are not directly associated with inflammatory responses.

   4. For the transcripts directly associated with FASN, do they contain predicted dsRNA-forming regions or known structured RNA elements? The current data do not sufficiently demonstrate whether these transcripts indeed form dsRNA structures. Although some are enriched in J2 immunoprecipitation, additional structural analyses, including computational prediction of dsRNA regions, would strengthen the conclusion.

   5. The authors should further investigate whether the enhanced antiviral activity observed upon FASN depletion is mediated by elevated endogenous dsRNA sensing. Are dsRNA sensors such as PKR, MDA5, or RIG-I more strongly activated in FASN knockout cells? Do these sensors exhibit increased binding to endogenous dsRNAs following FASN depletion?

   Significance

   Main strength: Uncover the proviral role of FASN via regulation of endogenous dsRNAs

   Limitations: Molecular mechanism on how FASN depletion increases the accessibility of dsRNAs without affecting their expression. Also, need better characterization of FASN-binding RNAs, especially their secondary structure.

   Read the original source
4. 

   Review Commons

   Jul 31, 2026

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This manuscript identifies a potential connection between FASN, endogenous dsRNAs, and innate immune signaling. The authors show that FASN depletion increases cytoplasmic dsRNA staining, enhances IFN transcription and restricts Sindbis virus replication. Overall, the basic observation seems to be solid and well-controlled. Depending on which journal this is being considered for, the central mechanism could be better developed and elucidated to entail more molecular details. As it currently stands, several important questions, especially ones regarding the FASN-RNA interactions remain unresolved.

   Major comments

   1. The conclusion that …

   Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

   Learn more at Review Commons

   ---

   Referee #1

   Evidence, reproducibility and clarity

   This manuscript identifies a potential connection between FASN, endogenous dsRNAs, and innate immune signaling. The authors show that FASN depletion increases cytoplasmic dsRNA staining, enhances IFN transcription and restricts Sindbis virus replication. Overall, the basic observation seems to be solid and well-controlled. Depending on which journal this is being considered for, the central mechanism could be better developed and elucidated to entail more molecular details. As it currently stands, several important questions, especially ones regarding the FASN-RNA interactions remain unresolved.

   Major comments

   1. The conclusion that FASN regulates dsRNA accessibility rather than dsRNA abundance requires further support. The authors show increased J2 staining without major changes in the global dsRNA landscape by J2-RIP-seq. However, alternative explanations could be: (1) changes in dsRNA structure or length distribution could affect J2 recognition, i.e. epitope masked by FASN binding; (2) differences in RNA-protein complex assembly could influence J2 accessibility; (3) Altered subcellular localization of dsRNAs could increase immunofluorescence signals without substantially affecting RIP-seq recovery.
   2. The nature of the FASN-RNA interaction remains unclear. The authors demonstrate that FASN associates with a subset of endogenous RNAs, but it is not clear whether this interaction is sequence-specific, structure-specific, or largely nonspecific. Are there common sequence motifs, secondary structures, repetitive elements, or specific transcript classes enriched among FASN-associated RNAs? Without a clearer understanding of RNA selectivity, it remains difficult to evaluate the biological significance of the observed interactions.
   3. The study would be significantly strengthened by complementation experiments. Although the FASN knockout phenotype is convincing, add-back experiments are needed to demonstrate specificity and probe potential mechanisms. Reconstitution with wild-type FASN should rescue the dsRNA and immune phenotypes. Furthermore, domain-specific mutants or catalytic mutants (e.g., palmitoylation) could help distinguish whether the observed effects depend on FASN enzymatic activity, a specific protein domain, or a non-canonical RNA-binding function. Such experiments would greatly improve the mechanistic depth of the study. It will also address whether the observed dsRNA increase phenotype is irreversible.
   4. The manuscript proposes that increased endogenous dsRNA sensing drives the inflammatory phenotype in FASN-deficient cells. However, direct evidence for enhanced PRR engagement is currently lacking. Although MDA5 and MAVS knockdown experiments suggest a possible contribution of this pathway, the observed knockdown effects are relatively modest and needs to be repeated by CRISPR knockout. Does FASN, MAVS double knockout cells rescue the slow growth phenotype? In addition, direct measurements of PRR activation, such as MDA5-RNA association, MAVS activation, IRF3 phosphorylation, or related downstream signaling events, were not examined.
   5. The antiviral phenotype observed during Sindbis virus infection is interesting, but the underlying mechanism remains uncertain. The authors propose that enhanced endogenous dsRNA sensing contributes to viral restriction, yet alternative explanations related to altered lipid metabolism, impaired membrane remodeling, or other consequences of FASN deficiency cannot be excluded. Especially when MDA5 and MAVS knockdown effect appears modest. Additional experiments disrupting interferon signaling or innate immune sensing pathways using the FASN, MAVS double knockout cells would help determine whether the antiviral phenotype is directly linked to the proposed dsRNA sensing mechanism.

   Significance

   This manuscript identifies a potential connection between FASN, endogenous dsRNAs, and innate immune signaling. The authors show that FASN depletion increases cytoplasmic dsRNA staining, enhances IFN transcription and restricts Sindbis virus replication. Overall, the basic observation seems to be solid and well-controlled. Depending on which journal this is being considered for, the central mechanism could be better developed and elucidated to entail more molecular details. As it currently stands, several important questions, especially ones regarding the FASN-RNA interactions remain unresolved.

   Read the original source
5. 

   Version published to 10.1101/2025.07.16.662511 on bioRxiv

   Jul 18, 2025