The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate
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Evaluation Summary:
In this study, DDX39B, a factor with known functions in mRNA splicing and nuclear export, is shown to regulate Foxp3, a lineage marker for T-regulatory cells in the immune system. The interactions are positioned in the context of multiple sclerosis and autoimmune inflammatory condition. The work would be of interest to immunologists and those studying RNA-mediated regulation and cellular signaling.
(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
Genes associated with increased susceptibility to multiple sclerosis (MS) have been identified, but their functions are incompletely understood. One of these genes codes for the RNA helicase DExD/H-Box Polypeptide 39B (DDX39B), which shows genetic and functional epistasis with interleukin-7 receptor-α gene ( IL7R ) in MS-risk. Based on evolutionary and functional arguments, we postulated that DDX39B enhances immune tolerance thereby decreasing MS risk. Consistent with such a role we show that DDX39B controls the expression of many MS susceptibility genes and important immune-related genes. Among these we identified Forkhead Box P3 ( FOXP3 ), which codes for the master transcriptional factor in CD4 + /CD25 + T regulatory cells. DDX39B knockdown led to loss of immune-regulatory and gain of immune-effector expression signatures. Splicing of FOXP3 introns, which belong to a previously unrecognized type of introns with C-rich polypyrimidine tracts, was exquisitely sensitive to DDX39B levels. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance.
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Author Response:
We thank the reviewers and editors for the thoughtful reviews and for the suggestion to use iTreg cells. We are currently setting up experiments with these cells to confirm the most important observations that were readily obtained in the Treg-like cell line MT-2 but difficult to test in primary Tregs. We look forward to resubmitting the manuscript that highlights the importance of post-transcriptional regulation of FOXP3 expression and its important links to autoimmune diseases.
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Evaluation Summary:
In this study, DDX39B, a factor with known functions in mRNA splicing and nuclear export, is shown to regulate Foxp3, a lineage marker for T-regulatory cells in the immune system. The interactions are positioned in the context of multiple sclerosis and autoimmune inflammatory condition. The work would be of interest to immunologists and those studying RNA-mediated regulation and cellular signaling.
(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):
DDX39B (also BAT1 and UAP56) has previously been shown to be a risk factor in Multiple Sclerosis, as well as other autoimmune diseases, and as a regulator of IL7 splicing. These data led the authors to postulate a broader role of DDX39B in immune regulation. They first carried out RNA-Seq from primary human CD4+ T cells depleted of DDX39B and found ~700 genes that are differentially expressed in a DDX39B-dependent manner, including about 10% of known MS-susceptibility genes detected. One of the most robustly identified DDX39B-sensitive genes in this experiment was the gene encoding the Fox3p transcription factor, a result that was reproduced in primary and cultured Treg cells. Consistently, loss of Fox3p target genes was also observed.
The authors go on to use RNA-Seq, subcellular fractionation and …
Reviewer #1 (Public Review):
DDX39B (also BAT1 and UAP56) has previously been shown to be a risk factor in Multiple Sclerosis, as well as other autoimmune diseases, and as a regulator of IL7 splicing. These data led the authors to postulate a broader role of DDX39B in immune regulation. They first carried out RNA-Seq from primary human CD4+ T cells depleted of DDX39B and found ~700 genes that are differentially expressed in a DDX39B-dependent manner, including about 10% of known MS-susceptibility genes detected. One of the most robustly identified DDX39B-sensitive genes in this experiment was the gene encoding the Fox3p transcription factor, a result that was reproduced in primary and cultured Treg cells. Consistently, loss of Fox3p target genes was also observed.
The authors go on to use RNA-Seq, subcellular fractionation and shRNA-resistant rescue experiments, conclude that reduced Fox3p expression in DDX39B-depleted cells is due to inefficient splicing of introns with C-rich polypyrimidine tracts. This is consistent with prior work suggesting a role of DDX39B in 3' splice site selection, although prior studies have not defined what specific role this protein plays. More broadly, the authors compare DDX39B-sensitive and resistant introns transcriptome-wide, and also find evidence for increased C-richness within DDX39B-dependent 3' splice sites - although they are careful to emphasize this sequence bias is not sufficient to confer DDX39B dependency.
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Reviewer #2 (Public Review):
In this manuscript, the authors have implicated the RNA helicase DDX39B, a gene associated with increased MS susceptibility, in Foxp3 splicing. In particular, the authors demonstrated that DDX39B is required to reduce Foxp3 intron retention, thereby promoting its expression, which taken together is expected to increase Treg functionality in the context of Multiple Sclerosis. The authors have used human total CD4 cells, the Treg-like cell line MT-2 and in some context, primary human Treg cells to perform these experiments. The biochemical experiments in my opinion are well done but primarily done in cell lines which include MT-2 as well as HEK293 and HeLa cells. Understandably, it is difficult to do these experiments in primary T cells, let alone Treg cells. However, at least some experiments with in vitro …
Reviewer #2 (Public Review):
In this manuscript, the authors have implicated the RNA helicase DDX39B, a gene associated with increased MS susceptibility, in Foxp3 splicing. In particular, the authors demonstrated that DDX39B is required to reduce Foxp3 intron retention, thereby promoting its expression, which taken together is expected to increase Treg functionality in the context of Multiple Sclerosis. The authors have used human total CD4 cells, the Treg-like cell line MT-2 and in some context, primary human Treg cells to perform these experiments. The biochemical experiments in my opinion are well done but primarily done in cell lines which include MT-2 as well as HEK293 and HeLa cells. Understandably, it is difficult to do these experiments in primary T cells, let alone Treg cells. However, at least some experiments with in vitro generated iTreg cells, along with functional experiments, would greatly strengthen the work.
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