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DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions

A Retraction to this article was published on 04 July 2018

A Corrigendum to this article was published on 20 January 2016

This article has been updated

Abstract

T helper 17 (TH17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective TH17 genes, and is required for TH17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp, which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5–RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5–RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in TH17-dependent diseases.

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Figure 1: Requirement for DDX5 in TH17 cytokine production in vitro and at steady state in vivo.
Figure 2: Role of DDX5 in mouse models of TH17-cell-mediated autoimmune disease.
Figure 3: Requirement for helicase-competent DDX5 and its associated lncRNA Rmrp in induction of TH17 cell cytokines.
Figure 4: Analysis of DDX5-dependent Rmrp function in TH17 cell differentiation.
Figure 5: Rmrp localization at RORγt-occupied genes and role in RORγt–DDX5 assembly.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

RNA-seq, TRAP-seq, RIP-seq, and ChIRP-seq data have been deposited in the Gene Expression Omnibus under accession number GSE70110.

Change history

  • 04 July 2018

    This Article has been retracted; see accompanying Retraction. Corrected online 20 January 2016. In this Article, author Frank Rigo was incorrectly listed with a middle initial; this has been corrected in the online versions of the paper.

  • 20 January 2016

    A Correction to this paper has been published: https://doi.org/10.1038/nature16968

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Acknowledgements

We thank M. V. Pokrovskii for unpublished ATAC-seq data and L. X. Garmire for suggestions on our manuscript. This work was supported by a Cancer Research Institute Irvington Postdoctoral Fellowship (W.H.), Institutional NRSA T32 CA009161_Levy (W.H.), National Multiple Sclerosis Society postdoctoral fellowship FG 2089-A-1 (L.W.), Career Development Award (329388) from the Crohn’s and Colitis Foundation of America (S.V.K.), Dale and Betty Frey Fellowship of the Damon Runyon Cancer Research Foundation 2105-12 (J.A.H.), HHMI Exceptional Research Opportunities Program (N.R.M. and N.H.), NIH F30 1F30CA189514-01 (R.A.F.), NIH P50-HG007735 and R01HG004361 (H.Y.C.), NIH R01AI080885 (D.R.L.), NIH R01DK103358 (R.B. and D.R.L.), and the Howard Hughes Medical Institute (H.Y.C. and D.R.L.).

Author information

Authors and Affiliations

Authors

Contributions

W.H. and D.R.L. designed experiments, analysed data and wrote the manuscript with input from the co-authors; B.T. and O.A. performed mass spectrometry studies; F.Ri. designed and synthesized control and Rmrp ASOs; S.J.G. and L.W. performed MOG-EAE immunization and blinded scoring; S.V.K. performed blinded histology scoring on colitis sections; W.H. and A.I.D. designed and performed ribosome TRAP-seq studies. S.M. and R.M.M. performed library preparation for RNA sequencing studies; N.R.M. and N.G.H. performed microscopy studies; F.Ra. provided recombinant full length His-tagged hRORγt, and F.V.F.-P. generated DDX5 conditional mutant animals. J.A.H. performed RORγt ChIP studies. C.P.N performed DDX5 studies in the thymus. R.A.F., W.H. and H.Y.C. performed ChIRP-seq experiments. E.R.M and R.B. performed statistical analyses on ChIRP-seq experiments.

Corresponding author

Correspondence to Dan R. Littman.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Identification of DDX5 as a RORγt-interacting partner.

a, Mass spectrometry experimental workflow. Sorted naive CD4+ T cells from wild-type mice were cultured in vitro in TH17-polarizing conditions for 48 h. Immunoprecipitation of endogenous RORγt was performed using RORγ/γt-specific antibodies on whole-cell lysates. RORγt enrichment in pull-down was confirmed by immunoblot. Immunoprecipitated proteins were digested and analysed by mass spectrometry. The listed DDX5 peptides were identified in the TH17 RORγt immunoprecipitate. b, Co-immunoprecipitaton of DDX5 with anti-RORγt in lysates of in vitro polarized TH17 cells. c, Cell surface phenotype of splenic and lymph node DAPICD4+CD8αCD19 T cells from wild-type and DDX5-T mice, examined by flow cytometry. d, Immunoblot of RORγt protein expression whole-cell lysate of cultured TH17 cells from wild-type or DDX5-T animals. For uncropped gels (b, d), see Supplementary Fig. 1. e, Immunofluorescence staining of RORγt in cultured TH17 cells from wild-type or DDX5-T mice. f, Immunofluorescence staining revealed nuclear localization of DDX5 in TH17 cells.

Extended Data Figure 2 DDX5 coregulates a subset of RORγt transcriptional targets in polarized TH17 cells.

a, Venn diagram of distinct and overlapping genes regulated by RORγt and/or DDX5, as determined from RNA-seq studies. b, Ingenuity Pathway Analysis (Qiagen) of DDX5- and RORγt-coregulated genes. c, IGV browser view showing biological replicate RNA-seq coverage tracks of control and DDX5-T from in vitro polarized TH17 cell samples at the Il17a, Il22, Ddx5 and Rorc loci. d, Independent RT–qPCR validation of RNA-seq results confirming effects of DDX5 deletion on RORγt target gene expression. Graphs show mean ± s.d.

Source data

Extended Data Figure 3 DDX5 chromatin localization in TH17 cells.

a, ChIP-seq-generated heatmap of DDX5 occupancy in regions centred on 16,003 RORγt-occupied sites (±2 kilobases (kb)). K-means linear normalization was used for clustering analysis by SeqMiner. Metagene analysis on cluster 1 depicts RORγt-occupied regions with DDX5 enrichment in wild-type but not DDX5-T cells; cluster 2 represents RORγt-occupied regions without DDX5 enrichment. b, IGV browser view of Il17a, Il17f and Rorc loci with ChIP-seq enrichment for RNA Pol II, RORγt and DDX5. c, Independent ChIP-qPCR of DDX5 in polarized TH17 cells. DDX5 occupancy at the Il17a and Il17f loci (as identified by RORγt ChIP-seq MACS peak called 32 and 39, respectively, from b) in control, DDX5-T or RORγt-deficient (RORγKO) cells. Results are representative of two independent experiments. Each experiment was performed with two technical replicates. Graph shows mean ± s.d. **P < 0.01 (unpaired, t-test).

Source data

Extended Data Figure 4 Influence of DDX5 on T-cell phenotypes in autoimmune disease models.

a, At 8 weeks after T-cell transfer, large intestine lamina propria mononuclear cells were evaluated for amounts of Il17a and Ifng mRNA by RT–qPCR. Results are representative of two independent experiments. Each experiment was performed using large intestines from three mice in each condition. RT–qPCR was performed with two technical replicates. Graph shows mean ± s.d. *P < 0.03 (unpaired, t-test). b, Gating strategy for analysis of TH17 and TH1 cells from large intestine of Rag2-deficient recipients of wild-type or DDX5-T naive T cells analysed at 8 weeks after T-cell transfer. c, Representative IL-17A and IFNγ intracellular staining of AquaCD4+RORγt+ TH17 cells in spinal cord of MOG-immunized animals on day 21.

Source data

Extended Data Figure 5 Noncoding RNAs enriched in DDX5 and RORγt RIP-seq studies.

a, DDX5-T cells were transduced with wild-type or helicase-mutant DDX5 and evaluated for DDX5 expression by immunofluorescence (left) and immunoblot (right) with anti-DDX5 antibody. For uncropped gels, see Supplementary Fig. 1. b, Venn diagram of noncoding RNAs detected by RIP-seq of ribosome-depleted TH17 cell lysates with anti-DDX5 and anti-RORγt antibodies. c, Abundance of top noncoding RNAs enriched in DDX5 and RORγt immunoprecipitates from polarized TH17 cell lysates depleted of ribosomes. Top, abundance of the noncoding RNAs in total lysate. d, RIP-qPCR experiments to compare Rmrp association with DDX5 in cultured TH17 and total thymocytes ex vivo. Results are representative of three independent experiments. Each experiment was performed with two technical replicates. Graph shows mean ± s.d. **P < 0.001 (unpaired, t-test).

Source data

Extended Data Figure 6 Rmrp and DDX5 knockdown in mouse and human TH17 cells.

a, RNA FISH analysis, using probes specific for Rmrp (green) and Malat1 (red) lncRNAs, in TH17 cells at 72 h after nucleofection with control (CTL) or Rmrp ASOs. b, Effect of Rmrp ASOs targeting different regions of Rmrp transcript on levels of Rmrp, Il17f and Ccr6 RNAs in polarized TH17 cells. c, Knockdown of DDX5 reduced IL-17A production in in vitro polarized human RORγt+ TH17 cells. **P < 0.01 (paired, t-test). Representative result shown in left panel. Each dot represents a different healthy donor (n = 4). Graphs show mean ± s.d.

Source data

Extended Data Figure 7 Effects of wild-type and mutant Rmrp in T-cell differentiation.

a, Il17a mRNA in cell lysates of in vitro polarized mouse TH17 cells at 96 h after transduction of control vector or wild-type Rmrp. Results are representative of two independent experiments. b, IFNγ production in polarized mouse TH1 cells at 96 h after transduction of control or Rmrp-encoding vector. Representative of two independent experiments. Each experiment was performed with two technical replicates. c, Comparison of human and mouse Rmrp sequences. Several mutations identified in CHH patients are highlighted. d, IL-17A production in polarized mouse TH17 cells at 96 h after transduction of wild-type or mutant Rmrp vectors. Representative of two independent experiments. e, Venn diagram depicting the number of distinct and overlapping genes regulated by RORγt, DDX5 and Rmrp in in vitro polarized TH17 cells. f, Expression of cytokine and Foxp3 mRNAs in T cells from wild-type or RmrpG270T/G270T mice cultured in vitro in TH17-, iTreg-, TH1- and TH2-polarizing conditions. Results are representative of two independent experiments. Each experiment was performed with two technical replicates. ***P < 0.001 (unpaired, t-test). g, ChIP-qPCR experiment using anti-RORγ/γt antibodies on chromatin of TH17 cells from wild-type or mutant mice cultured for 48 h in vitro. Each dot represents a different biological sample. Wild type, n = 2; RmrpG270T, n = 2. Results are representative of three separate independent experiments. Graphs show mean ± s.d. (unpaired, t-test).

Source data

Extended Data Figure 8 Effect of Ddx5 and Rmrp mutations in inflammation and thymocyte development.

a, Left, percentage weight change in Rag2−/− recipients of wild-type (black circles) or RmrpG270T/G270T (grey squares) naive CD4+ T cells in the transfer model of colitis. Animal weight was measured on day 56 (wild type, n = 8; RmrpG270T/G270T, n = 8, combined from three independent experiments). Graphs show mean ± s.d. ***P < 0.001 (unpaired, t-test). Middle, histology score (scale of 0–24) (wild type, n = 8; RmrpG270T/G270T, n = 5), combined from two independent experiments. **P < 0.01 (unpaired, t-test). Right, representative H&E staining of large intestine from Rag2−/− mice on day 56 after naive T-cell transfer. b, Mice with deletion of Ddx5 in early common lymphoid progenitors (DDX5-clpKO) have normal thymic development. Left, immunoblot of thymocyte lysates with anti-DDX5 antibody confirmed depletion of DDX5. Right, percentage of CD4 single-positive (SP), CD8α SP, double-positive (DP) and double-negative (DN) cells among total thymocytes. Each bar represents the result from one mouse (WT/het, n = 9; DDX5-clpKO, n = 6). For uncropped gels, see Supplementary Fig. 1. c, Thymocyte and peripheral T-cell surface phenotypes of wild-type and RmrpG270T/G270T knock-in mice at steady state. Peripheral T-cell gate, DAPI CD19CD8αCD4+.

Source data

Extended Data Figure 9 Association of Rmrp lncRNA with DDX5 and RORγt in vitro.

a, In vitro translated (TNT) HA-tagged wild-type or helicase-dead DDX5 and Flag-tagged RORγt were incubated with in vitro transcribed Rmrp. After capture on anti-HA or anti-Flag beads, the amount of lncRNA was determined by RT–qPCR. Data are representative of two independent experiments, and each experiment was performed with two technical replicates. b, Helicase requirement for in vitro interaction of DDX5 and RORγt. Recombinant GST–DDX5 (wild type or helicase-dead mutant) and His–RORγt full-length protein were synthesized in Escherichia coli, purified, and assayed for binding with or without in vitro transcribed Rmrp RNA in the presence exogenous ATP. c, Association of in vitro transcribed wild-type and mutant Rmrp with recombinant GST–DDX5 captured on glutathione beads (left) or with recombinant GST–DDX5 and His–RORγt captured with anti-His antibody. Amounts of associated Rmrp were quantified using RT–qPCR. Data are representative of two independent experiments. Each experiment was performed with two technical replicates. d, Comparison of ability of in vitro transcribed wild-type and RmrpG270T lncRNA to promote interaction between recombinant RORγt and DDX5 in vitro. All graphs show mean ± s.d. ***P < 0.001 (unpaired, t-test). For uncropped gels, see Supplementary Fig. 1.

Source data

Extended Data Figure 10 Rmrp chromatin localization in TH17 cells.

a, ChIRP-seq sample validation of Rmrp RNA pull-down over other nuclear noncoding RNAs using pools of ‘even’ or ‘odd’ capture probes. Graphs show mean ± s.d. b, ChIRP-qPCR of Rmrp RNA pull-down from wild-type TH17 cell lysate treated with or without RNase (n = 2). qPCR for each sample was performed with two technical replicates. Graph shows mean ± s.d. **P < 0.001 (unpaired, t-test). c, HOMER motif analysis reveals top three DNA motifs within Rmrp-enriched peaks. d, Significance of peak overlaps between Rmrp ChIRP-seq and ChIP-seq for BATF (n = 2), IRF4 (n = 7), STAT3 (n = 2), c-Maf (n = 2), RORγt (n = 2), CTCF (n = 2), RNA Pol II (n = 2), H3K27me3 (n = 4) and H3K4me3 (n = 3) in TH17 cells (hypergeometric distribution). Each dot represents a separate biological replicate of ChIP-seq experiments. e, Venn diagram depicting changes in peaks called from Rmrp (ChIRP-seq) experiments in wild-type and DDX5-T TH17 cells. f, Comparison of Rmrp chromatin occupancy (ChIRP-seq) at known RORγt occupied loci in in vitro polarized TH17 cells from wild-type and RmrpG270T/G270T mice.

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Huang, W., Thomas, B., Flynn, R. et al. DDX5 and its associated lncRNA Rmrp modulate TH17 cell effector functions. Nature 528, 517–522 (2015). https://doi.org/10.1038/nature16193

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  • DOI: https://doi.org/10.1038/nature16193

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