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The RNA-binding protein HuR is essential for the B cell antibody response

Nature Immunology volume 16, pages 415–425 (2015)Cite this article

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Abstract

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

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Figure 1: HuR expression is increased during B cell activation.
Figure 2: HuR-cKO mice have no major defects in B cell development.
Figure 3: B cells require HuR for responses to various classes of antigens.
Figure 4: Genes encoding molecules involved in energy metabolism are deregulated in HuR-deficient B cells.
Figure 5: HuR regulates intron use in B cells.
Figure 6: Alternative splicing of Dlst in HuR-cKO B cells diminished the enzymatic activity of α-KGDH.
Figure 7: The enzymatic activity of α-KGDH is required for the survival and proliferation of B cells.
Figure 8: ROS scavengers rescue HuR-deficient B cells from death.

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Acknowledgements

We thank K. Bates, D. Sanger, N. Evans, S. Walker, K. Tabbada, G. Morgan, R. Walker and the Babraham Institute Biological Support Unit for technical assistance. Supported by the Biotechnology and Biological Sciences Research Council (BBSRC BB/J001457/1 and BB/J00152X/1), the National Health and Medical Research Council of Australia (516786 to K.F.), the Victorian State Government Operational Infrastructure Support and Australian Government (National Health and Medical Research Council Independent Research Institute Infrastructure Support Scheme; K.F.), the European Molecular Biology Laboratory (EMBL Interdisciplinary Postdocs initiative fellowship to. K.Z.), the Russian Science Foundation (14-15-00133 to V.I.B. for work with the phosphonate analogs of 2-oxo acids) and the Slovenian Research Agency (J7-5460 for J.U. and T.C.).

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  1. Kirsten Fairfax

    Present address: Present address: The Department of Experimental Medicine, University of Melbourne, Parkville, Australia.,

Authors and Affiliations

  1. Laboratory of Lymphocyte Signalling and Development, The Babraham Institute, Cambridge, UK

    Manuel D Diaz-Muñoz, Sarah E Bell, Kirsten Fairfax, Elisa Monzon-Casanova & Martin Turner

  2. The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia

    Kirsten Fairfax

  3. MRC Centre for Immune Regulation, Institute of Microbiology and Infection, School of Immunity and Infection, University of Birmingham, Birmingham, UK

    Adam F Cunningham

  4. European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Trust Genome Campus, Cambridge, UK

    Mar Gonzalez-Porta

  5. Bioinformatics Group, The Babraham Institute, Cambridge, UK

    Simon R Andrews

  6. A. N. Belozersky Institute of PhysicoChemical Biology and Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia

    Victoria I Bunik

  7. Buchmann Institute for Molecular Life Sciences, Frankfurt, Germany

    Kathi Zarnack

  8. University of Ljubljana, Faculty of Computer and Information Science, Ljubljana, Slovenia

    Tomaž Curk

  9. Center for Molecular and Vascular Biology, KU Leuven, Belgium

    Ward A Heggermont & Stephane Heymans

  10. Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands

    Stephane Heymans

  11. Weill Cornell Medical College, Brain and Mind Research Institute, Burke Medical Research Institute, White Plains, New York, USA

    Gary E Gibson

  12. Division of Immunology, Biomedical Sciences Research Center “Alexander Fleming”, Vari, Greece

    Dimitris L Kontoyiannis

  13. Department of Genetics, UCL Genetics Institute, Environment and Evolution, University College London, London, UK

    Jernej Ule

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Contributions

M.D.D.-M., S.E.B., K.F. and M.T. designed and performed experiments; A.F.C. provided advice on experimental design; M.D.D.-M. and M.T. designed and performed all high-throughput sequencing experiments; M.D.D.-M., E.M.-C., M.G.-P., S.R.A. and K.Z. participated in bioinformatics analysis; T.C. and J.U. designed the iCount pipeline for iCLIP analysis; V.I.B. provided the inhibitors SP and PESP and advice on their cellular application and action; W.A.H. helped with procedures involving Dlst+/− mice; S.H. and G.E.G. provided Dlst+/− mice generated by Lexicon Pharmaceuticals; D.L.K. provided Elavl1tm1Dkon mice; and M.D.D.-M. and M.T. wrote the manuscript.

Corresponding author

Correspondence to Martin Turner.

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Integrated supplementary information

Supplementary Figure 1 HuR is required for GC formation.

(a) Proportion of BrdU+ Pre B cells in the bone marrow of Elavl1fl/flMb1+/+ (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) mice after 2.5 hours i.p. administration of BrdU (n=8 per group). (b) Analysis of BrdU+ FO and MZ B cells in the spleen of Ctrl and HuR-cKO mice. BrdU was administered orally during a 7-day period (n=5 per group). (c) Analysis of GC formation in littermate Elavl1fl/flMb1+/+ (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) mice immunized with SRBCs. Representative dot plots show gating strategy and frequency of GC B cells (B220+ GL7+ CD95+ cells). (d) Quantitation of the number of GC B cells in spleen (Mean ± SD, n=2 to 4 per genotype and time point).

Supplementary Figure 2 In vitro B cell activation is normal in the absence of HuR.

(a) Ca2+ signaling in Mb1-Cre (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) LN B cells following BCR activation using the anti-IgM antibody (B7.6) or the F(ab')2 anti-IgM antibody fragment. One of the three independent experiments performed is shown (n=3 biological replicates per group, data shown as mean value ± SEM). (b) Expression of CD69, CD86, CD25 and CD40 in Mb1-Cre (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) B cells after 48 hours in culture. Representative FACs plots are shown (6 biological replicates were analyzed per group in total). (c) Quantification of B cell activation markers in response to the indicated stimuli. Data is shown as MFI. Background staining levels in cells activated with LPS+IL4 were quantified using isotype antibodies (IC) (n=3 per group).

Supplementary Figure 3 mRNA abundance and translation of AU-rich element–binding proteins is not altered in the absence of HuR.

(a) (b) mRNA expression levels of a selected panel of AU-rich element binding proteins (AUBPs) in ex vivo (a) or LPS-activated (b) Elavl1fl/flMb1+/+ (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) B cells. The list of AUBPs annotated in MGI as protein binding to AU-rich elements (AmiGO 2 ID - GO:0017091) was completed with other well characterized AUBPs (Hnrnpc, Hnrnpd and Khsrp) and with all members of the Elavl- family of proteins. Normalized read counts (log2 scale) from 3-4 biological replicates are shown as a heat map. The fold change expression (HuR cKO vs WT) of each gene is shown as a bar plot. Padj values obtained after differential expression analysis using DESeq2 are shown. (c) (d) Analysis of ribosome footprints associated to the selected list of AUBPs. Data are shown as in a and b.

Supplementary Figure 4 Global change in the expression and translation of mRNAs involved in energy metabolism in the absence of HuR.

(a) Glycolysis (n=43), TCA Cycle (n=28) and Electron chain reaction (n=100) pathways are up regulated in GC B cells compared to naive B cells. RNAseq datasets from Hogenbirk MA et al. (PLoS One. 2013 Jul 29;8(7):e69815) (GEO ID – GSE47705) were analyzed and a Wilcoxon signed-rank test was performed to compare mRNA expression of a gene set versus all genes. Gene set definition was taken from Wikipathways. (b) ECDF representation of the mean mRNA expression of genes involved in Glycolysis, TCA Cycle and Electron chain reaction in ex vivo and in vitro activated splenic B cells. Wilcoxon signed-rank test was performed to compare mRNA expression in ex vivo B cells versus LPS- activated B cells. (c) ECDF and Wilcoxon signed-rank test were calculated using ribosome profiling data as in b. (d) Viability analysis of splenic B cells isolated from individual mice and activated with LPS+IL4 that were cultured in media alone (back line) or media with 2-deoxyglucose (1 mM 2DG, blue line), CDI-613 (100 µM, red line) or Oligomycin A (10 nM, grey line). The percentage and the number of live cells together with the mean cell size is shown at the indicated time points (mean ± SD; n=4 per group; one of the two independent experiments performed is shown, unpaired t test comparing non-treated versus treated cells at each time point; ***p<0.0005). (e) Proliferation analysis of splenic B cells cultured for 96 hours as in c. (f) Global analysis of the fold change (HuR-cKO/Ctrl) (log2) in mRNA expression and ribosome occupancy of those genes related to the energy metabolism (as defined in a). Data is shown as box whisker plot (Mean ± SD and 10-90 percentiles). A Wilcoxon signed-rank test comparing the data from Elavl1fl/flMb1+/+ (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) B cells in each condition (ex vivo B cells and LPS- activated B cells) was performed (*p<0.05, ***p<0.0005). (g) Glycolysis and Gluconeogenesis pathway. (h) TCA Cycle pathway. (i) Analysis of mRNA expression of Dlst and Elavl1 in naive and GC B cells (RNAseq data source: GEO ID – GSE47705). (j) Electron chain pathway (Source: Wikipathways). Differential expression analysis of ribosome profiling datasets was performed using DESeq2. Genes that were significantly increased in HuR cKO B cells are highlighted in orange in g, h and j. Dlst, highlighted in blue, was the only gene related to these pathways that was significantly decreased in HuR-cKO B cells.

Supplementary Figure 5 Analysis of HuR RNA targets in B cells.

(a) Unique HuR crosslink sites along the Dlst locus were visualized in the UCSC genome browser. Filtered data after peak binding analysis is shown as well as the original raw data. RefSeq gene and Ensembl predicted transcript tracks are included during visualization. (b) Magnification of the genomic area between exon 10 and exon 11. Two regions of interest (ROI 1 and ROI 2) are highlighted. (c) Detailed visualization of unique crosslink sites close to the 5’ splice site of intron 10 (ROI 1). (d) Annotation at single-nucleotide resolution of HuR crosslinks along the polypyrimidine tract near the alternative 3’ splicing site found in intron 10 (ROI 2). (e) Detection of immunopreciptated HuR-RNA complexes labelled with 32P-ATP during HuR iCLIP procedure. Representative image from one of the three independent experiments performed. The red rectangle indicates high molecular weight HuR-RNA complexes that were isolated for cDNA library preparation. (f) Summary of the number and percentage of cDNA molecules and crosslink sites that were uniquely identified in the three independent HuR iCLIP experiments performed. Data is shown associated to the mapping of single nucleotide crosslinks to genomic features (3’UTR, 5’UTR. ORF, intergenic or intron).

Supplementary Figure 6 HuR regulates intron use in B cells.

(a) Summary of intron-centric analysis of mRNAseq datasets. (b) Differential intron usage analysis. MA plot shows average mean expression versus fold change (Log2). Significant hits are highlighted in red (530 introns, padj = 0.1). (c) ECDF showing the mean expression of the 375 genes that have at least one differentially expressed intron. mRNAseq and ribosome profiling data obtained from Elavl1fl/flMb1+/+ (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) B cells treated in vitro with LPS for 48 hours. (d) Representative sashimi coverage plots generated in IGV of the indicated mRNAseq datasets. The different annotated transcript isoforms are indicated in blue. Arrows indicate alternative splicing events that differ in Ctrl. and HuR-cKO B cells. (e) Unique HuR crosslink sites along the Slc25a19 locus were visualized in the UCSC genome browser. Raw and filtered data after peak binding analysis (FDR<0.05) is shown. RefSeq packed annotation for Slc25a19 and transcripts annotated in Ensembl are included in the visualization.

Supplementary Figure 7 Alternative mRNA isoforms of Dlst are not translated into protein.

(a) Representative sashimi coverage plots generated in IGV of the indicated ribosome profiling datasets. No reads were mapped to alternative exon 10b in libraries from HuR-cKO B cells. (b) Amino acid sequence encoded by the wild type Dlst mRNA transcript ENSMUST00000053811 and predicted amino acid sequence encoded by the alternative Dlst transcript present in HuR-cKO B cells. Exon 10 and Exon 11 from Dlst transcript ENSMUST00000053811 are highlighted in yellow and blue respectively. Amino acid sequence after alternative exon 10b inclusion is highlighted in red. Asterisks in the sequence mark the stop codons. (c) Western blot analysis of Dlst expression in ex vivo and LPS- activated B cells from Elavl1fl/flMb1+/+ (Ctrl) and Elavl1fl/flMb1-Cre (HuR-cKO) mice. Two different antibodies generated using a synthetic human peptide from aa50 to aa100 (Abcam, ab187699) or a synthetic peptide from sequence around Val404 (Cell Signaling Technologies, Cat. No. 12618) were used. Representative immunoblots for Dlst, HuR and β-actin are shown. Molecular weight of wild- type Dlst protein is 49 KDa. Predicted molecular weight of truncated Dlst protein in HuR-cKO B cells is 27.8 KDa (not detected).

Supplementary Figure 8 Proliferation of HuR cKO B cells is restored by ROS scavengers.

In vitro analysis of HuR-cKO B cell proliferation in response to anti-IgM (B7.6 antibody, 10 µg/ml) and Baff (200 ng/ml) (a) and in response to anti-CD40 (10 µg/ml) + IL-4 (10 ng/ml) + IL-5 (5 ng/ml) (b). After cell staining with CellTrace™ Violet dye, B cells were cultured for 96 hours in complete RPMI media supplemented or not with sodium pyruvate (1 mM). Representative proliferation profiles are shown. Number of live cells in each generation was calculated based on CellTrace™ Violet dye dilution (Mean ± SD, n=3 per group).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 929 kb)

Supplementary Table 1

mRNAseq analysis (XLSX 4727 kb)

Supplementary Table 2

Ribosome profiling analysis (XLSX 3537 kb)

Supplementary Table 3

DEXSeq Exon centric analysis (XLSX 21237 kb)

Supplementary Table 4

DEXSeq Intron centric analysis (XLSX 17478 kb)

Supplementary Table 5

HuR iCLIP (mm10, FDR 0.05) (ZIP 13557 kb)

Supplementary Table 6

Reagents (XLSX 13 kb)

Supplementary Table 7

Antibodies (XLSX 12 kb)

Supplementary Table 8

Primers (XLSX 11 kb)

Supplementary Table 9 (XLSX 10 kb)

Source data

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Diaz-Muñoz, M., Bell, S., Fairfax, K. et al. The RNA-binding protein HuR is essential for the B cell antibody response. Nat Immunol 16, 415–425 (2015). https://doi.org/10.1038/ni.3115

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