ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity

  1. Michael J Moore
  2. Nathalie E Blachere
  3. John J Fak
  4. Christopher Y Park
  5. Kirsty Sawicka
  6. Salina Parveen
  7. Ilana Zucker-Scharff
  8. Bruno Moltedo
  9. Alexander Y Rudensky
  10. Robert B Darnell  Is a corresponding author
  1. Howard Hughes Medical Institute, The Rockefeller University, United States
  2. New York Genome Center, United States
  3. Howard Hughes Medical Institute, Ludwig Center at Memorial Sloan Kettering Cancer Center, United States
9 figures, 2 tables and 4 additional files

Figures

Figure 1 with 3 supplements
HITS-CLIP as a transcriptome-wide screen for ZFP36 function in T cells.

(A) Immunoblots with pan-ZFP36 antisera after activation of naïve CD4 +T cells in DC co-cultures, and with re-stimulation at day 3. Antibody and MW markers are shown on the left. NS* indicates a non-specific band. (B) Immunoblotting with pan-ZFP36 antisera 4 hr after activation of naïve CD4 +T cells, testing dependence on TCR stimulation (α-CD3), and co-stimulation (DCs or α-CD28). (C) ZFP36 HITS-CLIP design. (D) Representative autoradiogram of ZFP36 CLIP from activated CD4 +T cells using pan-ZFP36 antisera, with pre-immune and no-UV controls. Signal in Zfp36 KO cells is due to capture of ZFP36L1 RNP complexes. (E) The most enriched binding motifs and (F) annotation of binding sites from WT and Zfp36 KO cells. (G) Overlap of binding sites in WT and Zfp36 KO cells, stratified by peak height (PH). CLIP data are compilation of 4 experiments, with 3–5 total biological replicates were condition. (H) RNAseq in WT and Zfp36 KO CD4 +T cells activated under Th1 conditions for 4 hr. Log2-transformed fold-changes (KO/WT) are plotted as a cumulative distribution function (CDF), for mRNAs with 3’UTR, CDS, or no significant ZFP36 HITS-CLIP sites. Numbers of mRNAs in each category (n) and p-values from two-tailed Kolmogorov-Smirnov (KS) tests are shown. RNAseq data is a compilation of 2 experiments, with 3–4 biological replicates per condition.

https://doi.org/10.7554/eLife.33057.003
Figure 1—figure supplement 1
ZFP36 paralog expression in T cells.

(A) ZFP36 expression was measured by immunoblotting in a time course of CD8 +T -cell activation using pan-ZFP36 antisera. NS *indicates a presumed non-specific band. (B) 293T cells were transfected with indicated constructs expressing FLAG-HA (FH) tagged ZFP36 paralogs. Lysates were analyzed by immunoblotting with antibodies indicated below panels: anti-FLAG; two custom antisera (RF2046 and RF2047) raised against a C-terminal peptide of mouse ZFP36; commercial anti-ZFP36; and commercial anti-ZFP36L1/2. NS* indicates a presumed non-specific band. (C) Lysates from naïve or Th1-activated WT and Zfp36 KO CD4 +T cells were analyzed by immunoblotting with ZFP36- and ZFP36L1-specific antibodies. (D) Lysates from naïve, Th1-, or Th0-activated WT and Zfp36 KO CD4 +T cells (4 hr) were analyzed by immunoblotting with pan-anti-ZFP36. A representative blot is shown (left), with quantification across three independent experiments.

https://doi.org/10.7554/eLife.33057.004
Figure 1—figure supplement 2
ZFP36 HITS-CLIP.

(A) ZFP36 HITS-CLIP autoradiogram with a second pan-ZFP36 antisera (RF2046) is shown, with similar results to RF2047 (Figure 1D). (B) High MW and low MW ZFP36-RNA complexes were analyzed separately, yielding sites with very similar enriched motifs and transcript distribution. (C) Top three enriched motifs from HITS-CLIP sites in WT cells (left), Zfp36 KO cells (middle), and sites only identified in Zfp36 KO cells (right). Correspondence to previously known motifs is shown. (D) Motif analysis of ZFP36 HITS-CLIP sites from WT cells in 3’UTR (left), and the position of ZFP36 binding sites in 3’UTR depicted as a coverage map (right). (E) Motif analysis of ZFP36 HITS-CLIP sites from WT cells in CDS (left), and representative CDS binding sites visualized in UCSC Genome Browser. CLIP reads supporting CDS binding sites span exon-intron boundaries, consistent with 5’-ss motif enrichment. Cross-link-induced truncations (CITS) are shown, confirming that binding occurs within the coding exon.

https://doi.org/10.7554/eLife.33057.005
Figure 1—figure supplement 3
Regulation of mRNA abundance by ZFP36.

(A) Transcriptome profiling data plotted as RPKM in Zfp36 KO cells versus RPKM in WT cells. Few large changes were observed, with the exception of highlighted mRNAs (e.g. Ig mRNAs) that were silent in WT cells, but show expression in KO cells. The absence of CLIP binding sites and ZFP36 consensus motifs in this subset suggests secondary effects. (B) Transcriptome profiling results are depicted as a CDF as in Figure 1 for sites further supported by CITS. (C) Transcriptome profiling results are depicted as a CDF as in Figure 1, further stratified by the magnitude of ZFP36 binding in 3’UTR. 3’UTR target mRNAs overall (violet) show a significant shift relative to non-targets (black), but the top 20% of sites defined by peak height (red) show no significant shift. Numbers of mRNAs in each category (n) and p-values from two-tailed Kolmogorov-Smirnov (KS) tests are shown. (D) Analysis as in (C) for mRNAs with ZFP36 binding in CDS.

https://doi.org/10.7554/eLife.33057.006
Figure 2 with 1 supplement
ZFP36 regulates target protein levels in T cells.

(A) Levels of mRNA and protein in Zfp36 KO and WT T cells and ZFP36 CLIP tracks measured 4 hr post-activation for targets with robust 3’UTR ZFP36 binding. RNA values are mean RPKM ± S.E.M. of 4 biological replicates. Protein values are mean fluorescence intensities (MFI) ± S.E.M. for 3–4 mice per condition. (B) Design of GFP reporters with WT 3’UTR (WT-UTR) or one lacking the ZFP36 binding site (Δ-UTR). (C) WT-UTR or Δ-UTR reporters were co-transfected into 293 cells with Zfp36 (+) or vector alone (-). 24 hr post-transfection, reporter mRNA and protein levels were measured by RT-qPCR and flow cytometry, respectively. Values are mean ± S.D. of 4 biological replicates in each condition. Data for Ifng reporters show one representative experiment of three performed. Tnf and Cd69 reporters were analyzed in one experiment. Results of two-tailed t-tests: *=p < 0.05; **=p < 0.01; ***=p < 0.001.

https://doi.org/10.7554/eLife.33057.007
Figure 2—figure supplement 1
Protein levels of ZFP36 CLIP targets.

Representative flow cytometry data for analyses in Figure 2A.

https://doi.org/10.7554/eLife.33057.008
Figure 3 with 1 supplement
ZFP36 regulates target ribosome association.

(A) Ribosome profiling of Zfp36 KO and WT CD4 +T cells. (B) Changes in ribosome association between Zfp36 KO and WT cells plotted as a CDF. (C) Change in translation efficiency (ΔTE) between Zfp36 KO and WT was calculated as a delta between log2(KO/WT) from ribosome profiling and RNAseq datasets. The distribution of ZFP36 targets in mRNAs ranks by ΔTE is shown (left), along with normalized enrichment scores and FDRs from GSEA (right). Intron-bound mRNAs are shown as a representative gene set that show no enhanced TE in Zfp36 KO cells. (D) Normalized coverage of ribosome profiling reads for Tnf and Ifng mRNAs in Zfp36 KO and WT cells, with p-values from binomial tests. (E) Normalized coverage of ribosome profiling reads across all mRNAs for Zfp36 KO and WT cells. Ribosome profiling data are a compilation of two experiments, with four total biological replicates per conditions.

https://doi.org/10.7554/eLife.33057.009
Figure 3—figure supplement 1
Analysis of ZFP36 translational control by ribosome profiling.

(A) Outline of biochemical strategy to isolate ribosome-protected fragments (RPFs), corresponding to ribosome associated mRNAs. (B) Normalized RPF read coverage is shown for ZFP36 mRNA in WT and Zfp36 KO cells. RPF coverage is lost in Zfp36 KO cells downstream of the site of gene disruption (Taylor et al., 1996), confirming our biochemical strategy faithfully identifies translating mRNAs. (C) Analysis of shifts in ribosome association is shown as in Figure 3B, further stratified by the magnitude of ZFP36 binding. The top 20% of sites (red) show similar shifts to sites overall (violet) for both 3’UTR (top panel) and CDS (bottom panel) in ribosome profiling experiments.

https://doi.org/10.7554/eLife.33057.010
Figure 4 with 2 supplements
ZFP36 regulates T-cell activation kinetics.

(A) Gene expression patterns from a T-cell activation time course (Yosef et al., 2013) were partitioned by k-means, and enrichment of ZFP36 3’UTR and CDS targets was determined across clusters (Fisher’s Exact Test). Mean expression of genes in the three clusters most enriched (left) or depleted (right) for ZFP36 targets is plotted. (B) Enriched GO terms among ZFP36 HITS-CLIP targets (full results in Supplementary File 2). (C) Proliferation of naïve CD4 +Zfp36 KO and WT T cells in the indicated time windows after activation, measured by 3H-thymidine incorporation (D) Fractions of apoptotic annexin-V+ and (E) proliferating Ki67 +CD4+T cells 24 hr post-activation. Mean ± S.E.M. is shown; circles are individual mice (n = 3–4 per genotype). (F) Proliferation of BG2 TCR-transgenic CD4 +T cells cultured with DCs pulsed with cognate (β-gal) or irrelevant (OVA) peptide. Mean ± S.E.M. is shown (n = 5 mice per genotype). (G) Proliferation of CD4 +T cells co-cultured with syngeneic (C57BL6/J) or allogeneic (Balb-c) DCs. Mean ± S.E.M. of three replicate cultures is shown. (H) Levels of CD69 and CD25 after activation of Zfp36 KO and WT naïve CD4 +T cells. Mean ± S.E.M. is shown (n = 3–4 mice per genotype). (I) Naïve and effector subsets 40 hr post-activation in Zfp36 KO and WT CD4 +T cells. Representative plots are shown (top), along with mean ± S.E.M and circles for individual mice (n = 4 per genotype). For (C–I), results of two-tailed t-tests: *=p < 0.05; **=p < 0.01; ***=p < 0.001. Data are representative of three (H) or two (C–G, I) independent experiments.

https://doi.org/10.7554/eLife.33057.011
Figure 4—figure supplement 1
ZFP36 targets regulate T-cell activation.

The T cell activation KEGG pathway is shown with robust ZFP36 CLIP targets shaded based on the location and timing of ZFP36 binding.

https://doi.org/10.7554/eLife.33057.012
Figure 4—figure supplement 2
ZFP36 regulates early activation across T cell lineages.

(A) Measurement of proliferation after activation of CD8 +T cells, varying TCR signal strengths (anti-CD3); co-stimulation (DCs); and the presence of recombinant IL-2. The mean c.p.m. ± S.E.M. of three replicate cultures per condition is shown. (B) Measurement of proliferation by thymidine incorporation after activation of naïve CD4 +T cells is shown in the presence or absence of excess recombinant IL-2; the presence of neutralizing IL-2 antibodies; and (C) under various Th skewing conditions. Mean ± S.E.M. is shown, with circles indicating individual mice (n = 3–4 per genotype). For (A–C), results of two-tailed t-tests are shown above relevant comparisons: *=p < 0.05; **=p < 0.01; ***=p < 0.001. One representative experiment of two performed is shown.

https://doi.org/10.7554/eLife.33057.013
ZFP36 regulation of T cell activation kinetics cell-intrinsic.

(A) Lethally irradiated mice were reconstituted with congenically marked WT and Zfp36 KO BM to generate mixed chimeras. 10–12 weeks after reconstitution, naïve CD4 +WT and Zfp36 KO T cells were sorted, then activated ex vivo separately or mixed 1:1. (B) Proliferating Ki67 +cells were measured 24 hr after activating naïve CD4 +T cells under Th0 or Th1 conditions. (C) Cultures with a 1:1 starting ratio of naïve WT and Zfp36 KO CD4 +T cells were examined 3 days post-activation. Data from one experiment of two performed are shown.

https://doi.org/10.7554/eLife.33057.014
Figure 6 with 2 supplements
Accelerated signs of in vitro T cell exhaustion in absence of ZFP36.

(A) Log2-transformed RPKM values from Zfp36 KO versus WT CD4 +Th1 cell RNAseq 72 hr post-activation, with red indicating differential expression (FDR < 0.05). Lines mark 2-fold changes. RNAseq data represent one experiment with three biological replicates per condition. (B) Log2-transformed fold-changes (KO/WT) plotted as a CDF, for mRNAs with 3’UTR, CDS, or no significant ZFP36 HITS-CLIP. Numbers of mRNAs in each category (n) and p-values from KS tests are indicated. (C) The gene expression profile in Zfp36 KO CD4 +T cells 72 hr post-activation was compared to reported profiles of CD4 +T cell exhaustion using GSEA. Upregulated (orange) and downregulated (gray) gene sets in exhausted T cells showed strong overlap with corresponding sets from Zfp36 KO T cells (FDR < 0.001, hypergeometric test). (D) IFN-γ and TNF-α measured by ICS 3 and 5 days after activation of naïve CD4 +T cells. (E) IFN-γ and TNF-α in culture supernatants 3 and 5 days after activation of naïve CD4 +T cells. (F) PD-1, ICOS, and LAG-3 expression 5 and 13 days after activation under Th0 or Th1 conditions. (D–F) show mean ± S.E.M.; circles are individual mice (n = 3–5 per genotype). (G) Measurements as in (F) for Zfp36 KO and WT CD4 +T cells derived from mixed BM chimeras. Cells were activated under Th1 conditions for 13 days, either separately or mixed 1:1. For (D–G), one representative experiment of two performed is shown. Results of two-tailed t-tests: *=p < 0.05; **=p < 0.01; ***=p < 0.001; ****=p < 0.0001.

https://doi.org/10.7554/eLife.33057.015
Figure 6—figure supplement 1
Analysis of ZFP36 function 3 days after T cell activation.

(A) Top enriched motifs and (B) annotation of ZFP36 HITS-CLIP sites from CD4 +T cells activated for 3 days under Th1 conditions. Site distribution was similar to the 4 hr time point, except for increased intronic binding, which may reflect increased nuclear permeability in these rapidly cycling cultures.

https://doi.org/10.7554/eLife.33057.016
Figure 6—figure supplement 2
Dysfunction of Zfp36 KO T cells at late time points.

(A) GSEA analysis found that genes promoting proliferation and cell division, including transcriptional targets of E2F and Myc, were down-regulated in Zfp36 KO Th1 cells versus WT 3 days after activation. Enrichment score distributions are plotted, with the distribution of interrogates genes shown below. (B) WT and Zfp36 KO CD4 +T cells sorted from mixed BM chimeras were analyzed for PD-1 and ICOS expression after long-term (13 days) activation under Th1 conditions. Separated cultures of WT and Zfp36 KO confirmed differential expression of these receptors, but expression was similar in 1:1 ‘re-mixed’ cultures. Adding recombinant IFN-γat 20 ng/ml (similar to levels measured in KO culture supernatants) did not shift PD-1 expression in WT cells toward levels observed in Zfp36 KO, but did so for ICOS expression. Data from one representative experiment of two are shown.

https://doi.org/10.7554/eLife.33057.017
Figure 7 with 3 supplements
ZFP36 regulates anti-viral immunity.

(A) Virus-specific CD4 + or (B) CD8 +T cells were tracked in peripheral blood using MHC-tetramers after LCMV Armstrong infection (n = 8–9 mice per genotype). (C) Virus-specific CD4 +T cells and CD69 expression on CD4 +T cells in peripheral blood at early time points post-infection (p.i.) (n = 7–8 mice per genotype). (D) Virus-specific CD8 +T cells and CD69 expression on CD8 +T cells in peripheral blood at early time points p.i. (n = 7–8 mice per genotype). (E) Virus-specific CD4 +and (F) CD8 +T cells in spleen after LCMV infection (n = 5–8 mice per genotype). (G) Fraction of CD4 +T cells producing IFN-γ and TNF-α in splenic CD4 +T cells 6 days p.i., after ex vivo stimulation with GP66-77 peptide (n = 7–8 mice per genotype). (H) Levels of IFN-γ and TNF-α(gated on cytokine-producing CD4 +cells) 6 days p.i. after ex vivo stimulation with GP66-77 (n = 7–8 mice per genotype). (I) Raw percentage of bifunctional IFN-γ+TNF-α+CD4+cells in spleen 6 days p.i. after ex vivo stimulation with GP66-77 (left), or normalized to percentage of GP66-77 tetramer +cells (n = 7–8 mice per genotype). (J) Levels of LCMV genomic RNA in spleen measured by RT-qPCR (n = 9–14 per group). For (A–J), mean values ± S.E.M. are shown, with circles as individual mice. Results of two-tailed t-tests: *=p < 0.05; **=p < 0.01; ***=p < 0.001; ****=p < 0.0001. In each panel, one representative experiment of two is shown.

https://doi.org/10.7554/eLife.33057.018
Figure 7—figure supplement 1
The T cell compartment in naïve Zfp36 KO mice is largely normal.

(A) Counts of CD4 +and CD8+T cells in peripheral blood of WT and Zfp36 KO mice (n = 9–10 per genotype). Mean values ± S.E.M are shown; circles are individual mice (n = 9–10 per genotype) (B) Counts of thymocytes and distribution among T cell development stages in WT and Zfp36 KO mice (n = 11 per genotype; DN = double negative; DP = double positive; CD4-SP = CD4 single positive; CD8-SP = CD8 single positive) (C) Counts of CD4 + and CD8+T cells in spleen or (D) as a proportion of splenocytes in WT and Zfp36 KO mice (n = 5 mice per genotype). (E) Percentages of naïve CD4 +and CD8+T cells in spleen, defined as CD25-CD62L-hiCD44-lo (n = 11 per genotype). For (A–E), mean values ± S.E.M are shown with circles as individual mice. (F) Percentages of CD4 +CD25 hi cells in spleen (n = 11 per genotype). For (A–F), mean values ± S.E.M are shown with circles as individual mice. (G) Percentages of FoxP3 +iTreg cells from WT and Zfp36 KO FoxP3-GFP transgenic mice indicated skewing conditions (mean ±S.E.M. is shown for n = 2 mice per genotype). (H) Percentages of CD4 + and CD8+cells producing the indicated cytokines by intracellular flow cytometry, following 5 hr of PMA/ionomycin stimulation of splenocytes directly ex vivo (mean ± S.E.M. is shown for n = 6 per genotype). (I) Percentages of CD4 +T cells producing the indicated lineage-specific effector cytokines is shown under various skewing conditions (mean ± S.E.M. is shown for n = 3 mice per genotype). For (A–I) results of two-tailed t-tests are shown beneath relevant panels when significant differences were observed: *=p < 0.05. Otherwise, differences were not significant.

https://doi.org/10.7554/eLife.33057.019
Figure 7—figure supplement 2
ZFP36 regulates anti-viral immunity.

(A) The fraction of CD8 +T cells producing IFN-γ and IFN-γ proteins levels were measured splenic CD8 +T cells by ICS 6 days post-infection, after ex vivo stimulation with the LCMV antigenic peptide GP33-41 (n = 5–7 per genotype). (B) The fraction of CD8 +T cells producing TNF-α and TNF-α proteins levels were measured in splenic CD8 +T cells by ICS 6 days post-infection, after ex vivo stimulation with the LCMV antigenic peptide GP33-41 (n = 5–7 per genotype). (C) The raw percentage of bifunctional IFN-γ+TNF-α+CD8+cells in spleen 6 days post-infection after ex vivo stimulation with GP33-41 (left), or normalized to percentage of GP33-41 tetramer +cells (right) (n = 5–7 per genotype). (D) Plots depicting the relationship between LCMV load and levels of tetramer +virus specific T cells in spleens of WT and Zfp36 KO animals, 6 days post-infection. R-squared and p-values are shown for linear regression analysis. For (A–D), mean values ± S.E.M are shown with circles as individual mice. Results of two-tailed t-tests are shown beneath relevant panels when significant differences were observed: *=p < 0.05; **=p < 0.01; ***=p < 0.001, ****=p < 0.0001. Data from one representative experiment of two are shown.

https://doi.org/10.7554/eLife.33057.020
Figure 7—figure supplement 3
LCMV infection in mixed bone marrow chimeras.

(A) Schematic of mixed chimeric experiments. (B) Analysis of T cell numbers in peripheral blood in mixed bone marrow chimeras 10 weeks after constitution. Values were normalized to levels of Thy1.1 WT T cells, and the mean ± S.D. are shown (n = 15 mice). (C) Levels of LCMV-specific CD4 + and (D) CD8 +T cells were determined in peripheral blood by MHC-tetramer staining over a time course of LCMV Armstrong infection. Values are mean ± S.D (n = 15 mice). Data are shown for one representative experiment. A second experiment analyzing d0-d10 showed similar results.

https://doi.org/10.7554/eLife.33057.021

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Zfp36NAEntrez ID: 22695
Gene (M. musculus)Zfp36l1NAEntrez ID: 12192
Gene (M. musculus)Zfp36l2NAEntrez ID: 12193
Strain (M. musculus),
strain background
(C57BL6/J)
C57BL6/JJackson LaboratoryStock No: 000664
Strain (M. musculus),
strain background
(C57BL6/J)
ZFP36 KOPMID:8630730gift from P. Blackshear
Strain (M. musculus),
strain background
(C57BL6/J)
BG2PMID:19478869gift from N. Restifo
Strain (M. musculus),
strain background
(C57BL6/J)
Thy1.1Jackson LaboratoryStock No: 000406
Strain (M. musculus),
strain background
(C57BL6/J)
CD45.1Jackson LaboratoryStock No: 002014
Strain (M. musculus),
strain background
(C57BL6/J)
FoxP3-EGFPJackson LaboratoryStock No: 006769
Strain (Lymphocytic
Choriomeningitis Virus,
LCMV), strain background
(Armstrong)
LCMV ArmPMID:6875516
Cell line (Homo sapien)293 T-rexLife TechnologiesCat# R71007
Cell line (H. sapien)293TATCCATCC Cat# CRL-3216,
RRID:CVCL_0063
Cell line (M. musculus)J558L/GM-CSFPMID:1460426
Transfected construct
(M. musculus)
pOZ-N-FH-ZFP36This studymouse Zfp36 ORF in
pOZ-N vector
Transfected construct
(M. musculus)
pOZ-N-FH-ZFP36L1This studymouse Zfp36l1 ORF in
pOZ-N vector
Transfected construct
(M. musculus)
pOZ-N-FH-ZFP36L2This studymouse Zfp36l2 ORF in
pOZ-N vector
Transfected constructpOZ-N-FH vectorPMID:14712665
Transfected constructpcDNA3.1(+)Life TechnologiesCat# V79020
Transfected constructpcDNA3.1(+)-Acgfp1-
IFNG-WT-UTR
This paperAcgfp1 with mouse Ifng
3'UTR
Transfected constructpcDNA3.1(+)-Acgfp1-
IFNG-WT-UTR
This paperAcgfp1 with mouse Ifng
3'UTR with Zfp36 binding
site deleted
Transfected constructpcDNA5/FRT/TOLife TechnologiesCat# V652020
Transfected constructpcDNA5/FRT/TO/
Acgfp1-TNF-WT-UTR
This paperAcgfp1 with mouse Tnf 3'UTR
Transfected constructpcDNA5/FRT/TO/
Acgfp1-TNF-Δ-UTR
This paperAcgfp1 with mouse Tnf 3'UTR
with Zfp36 binding site deleted
Transfected constructpcDNA5/FRT/TO/
Acgfp1-CD69-WT-UTR
This paperAcgfp1 with mouse Cd69 3'UTR
Transfected constructpcDNA5/FRT/TO/
Acgfp1-CD69-Δ-UTR
This paperAcgfp1 with mouse Cd69 3'UTR
with Zfp36 binding site deleted
AntibodyRabbit anti-pan-
ZFP36 RF2046
This paperCovance custom service1:2000 for Western
AntibodyRabbit anti-pan-
ZFP36 RF2047
This paperCovance custom service1:2000 for Western
Antibodyanti-Br-dUMilliporeMillipore Cat# MAB3222;
RRID:AB_11212494
5 μg per IP
Antibodyrabbit anti-TTP/ZFP36SigmaSigma-Aldrich Cat# T5327;
RRID:AB_1841222
1:500
Antibodyrabbit anti-ZFP36L1/2
(BRF1/2)
CSTCell Signaling Technology
Cat# 2119S;
RRID:AB_10695874
1:500
Antibodymouse anti-FLAGSigmaSigma-Aldrich Cat# F3165;
RRID:AB_259529
1:500
Antibodymouse anti-FUSSanta CruzSanta Cruz Biotechnology
Cat# sc-47711;
RRID:AB_2105208
1:1000
Antibodyrabbit anti-FUSNovusNovus Cat# NB100-562;
RRID:AB_10002858
1:10000
antibodygoat anti-rabbit-IgG-
680RD
LICORLI-COR Biosciences
Cat# 925–68071;
RRID:AB_2721181
1:25000
Antibodygoat anti-rabbit-IgG-
800CW
LICORLI-COR Biosciences
Cat# 925–32211;
RRID:AB_2651127
1:25000
Antibodygoat anti-mouse-IgG-
800CW
LICORLI-COR Biosciences
Cat# 925–32210;
RRID:AB_2687825
1:25000
Antibodyanti-CD4-PerCP-Cy5.5BD BiosciencesBD Biosciences Cat# 550954;
RRID:AB_393977
1:400
Antibodyanti-CD25-PE/Cy7BiolegendBioLegend Cat# 102016;
RRID:AB_312865
1:400
Antibodyanti-CD62L-APCBD BiosciencesBD Biosciences Cat# 561919;
RRID:AB_10895379
1:800
Antibodyanti-CD44-PEBD BiosciencesBD Biosciences Cat# 560569;
RRID:AB_1727484
1:1000
Antibodyanti-CD8-BV510BiolegendBioLegend Cat# 100752;
RRID:AB_2563057
1:400
Antibodyanti-Thy1.2-BUV395BD BiosciencesBD Biosciences Cat# 5652571:200
Antibodyanti-Thy1.1-FITCBiolegendBioLegend Cat# 202504;
RRID:AB_1595653
1:400
Antibodyanti-CD19-eFlour780eBiosciencesThermo Fisher Scientific
Cat# 47-0193-82;
RRID:AB_10853189
1:200
Antibodyanti-CD11b-eFlour780eBiosciencesThermo Fisher Scientific
Cat# 47-0112-82;
RRID:AB_1603193
1:200
Antibodyanti-CD11c-eFlour780eBiosciencesThermo Fisher Scientific
Cat# 47-0114-80;
RRID:AB_1548663
1:100
Antibodyanti-NK1.1-eFlour780eBiosciencesThermo Fisher Scientific
Cat# 47–5941;
RRID:AB_10853969
1:100
Antibodyanti-CD69-FITCBiolegendBioLegend Cat# 104506;
RRID:AB_313109
1:200
Antibodyanti-BCL2-PE/Cy7BiolegendBioLegend Cat# 633512;
RRID:AB_2565247
1:200
Antibodyanti-TNF-APC/Cy7BD BiosciencesBD Biosciences Cat# 560658;
RRID:AB_1727577
1:200
Antibodyanti-IFNG-Alexa647BD BiosciencesBD Biosciences Cat# 557735;
RRID:AB_396843
1:1000
Antibodyanti-Ki67-PE/Cy7BiolegendBioLegend Cat# 652426;
RRID:AB_2632694
1:200
Antibodyanti-CD44-BUV737BD BiosciencesBD Biosciences Cat# 5643921:400
Antibodyanti-PD-1-PE/Cy7BiolegendBioLegend Cat# 135216;
RRID:AB_10689635
1:200
Antibodyanti-LAG3-APCBiolegendBioLegend Cat# 125210;
RRID:AB_10639727
1:200
Antibodyanti-ICOS-PEBiolegendBioLegend Cat# 107706;
RRID:AB_313335
1:200
Antibodyanti-mouse-IL-2
(neutralizing)
BiolegendBioLegend Cat# 503705;
RRID:AB_11150768
10 μg/ml
Antibodyanti-mouse-CD3e
(stim)
BiolegendBioLegend Cat# 100314;
RRID:AB_312679
0.25 μg/ml
Antibodyanti-mouse-CD28
(co-stim)
BiolegendBioLegend Cat# 102112;
RRID:AB_312877
1 μg/ml
Other, MHC tetramerLCMV GP33-41-specific
H-2Db- MHC-tetramer,
PE conjugate
MBLCat# TS-M512-11:400
Other, MHC tetramerLCMV GP66-77-specific
I-Ab- MHC-tetramer,
APC conjugate
NIH Tetramer Core
Facility
1:300
Peptide, recombinant
protein
LCMV GP33-41 peptide
KAVYNFATM
Life TechnologiesCustom synthesis
Peptide, recombinant
protein
LCMV GP66-77 peptide
DIYKGVYQFKSV
Life TechnologiesCustom synthesis
Peptide, recombinant
protein
Ovalbumin p257
peptide SIINFEKL
Life TechnologiesCustom synthesis
Peptide, recombinant
protein
Ovalbumin p323 peptide
ISQAVHAAHAEINEAGR
Life TechnologiesCustom synthesis
Peptide, recombinant
protein
Recombinant mouse
TNF-α
R and D SystemsCat# 410-MT-050
Peptide, recombinant
protein
Recombinant human
IL-2
PeprotechCat# 200–02
Peptide, recombinant
protein
Recombinant mouse
IL-12
eBiosciencesCat# 14-8121-80
Peptide, recombinant
protein
Recombinant mouse
IL-23
eBiosciencesCat# 14-8231-63
Peptide, recombinant
protein
Recombinant mouse
IL-6
eBiosciencesCat# 14–8061
Peptide, recombinant
protein
Recombinant human
TGF-β1
R and D SystemsCat# 240-B-010
Peptide, recombinant
protein
T4 Polynucleotide
Kinase
New England BiolabsCat# M0201L
Peptide, recombinant
protein
T4 RNA ligase 2,
truncated KQ
New England BiolabsCat# M0373L
Peptide, recombinant
protein
CircLigaseEpicentreCat# CL4115K
Peptide, recombinant
protein
Phusion PolymeraseNew England BiolabsCat# M0530L
Peptide, recombinant
protein
Micrococcal nucleaseNew England BiolabsCat# M0247S
Peptide, recombinant
protein
RNAsin PlusPromegaCat# N2611
Peptide, recombinant
protein
RNAse AAffymetrixCat# 70194Y
Peptide, recombinant
protein
RNAse IThermo FisherCat# EN0601
Peptide, recombinant
protein
alkaline phosphataseRocheCat# 10 713 023 001
Commercial assay or
kit
Xtremegene 9
Transfection Reagent
RocheCat# 06 365 787 001
Commercial assay or
kit
Mouse CD4
microbeads
MiltenyiCat# 130-049-201
Commercial assay or
kit
Mouse CD8
microbeads
MiltenyiCat# 130-049-401
Commercial assay or
kit
Mouse CD11c
microbeads
MiltenyiCat# 130-108-338
Commercial assay or
kit
Mouse CD19
microbeads
MiltenyiCat# 130-052-201
Commercial assay or
kit
Mouse CD11b
microbeads
MiltenyiCat# 130-049-601
Commercial assay or
kit
Mouse CD4 + CD62L +
T cell isolation kit
MiltenyiCat# 130-093-227
Commercial assay or
kit
Trizol ReagentLife TechnologiesCat# 15596026
Commercial assay or
kit
High Pure RNA
Isolation Kit
RocheCat# 11828665001
Commercial assay or
kit
Truseq RNA Library
Kit
IlluminaCat# RS-122–2001
Commercial assay or
kit
Ribo-Zero rRNA
removal kit
IlluminaCat# MRZH11124
Commercial assay or
kit
Cytofix/Cytoperm
Kit
BD BiosciencesCat# 554722
Commercial assay or
kit
Ampure XP beadsBeckman-CoulterCat# A63881
Commercial assay or
kit
Quant-IT dsDNA Assay
Kit, High Sensitivity
Life TechnologiesCat# Q33120
Commercial assay or
kit
iQ SYBR Green SuperMixBioradCat# 1708880
Commercial assay or
kit
iScript cDNA Synthesis
Kit
BioradCat# 1708891
Chemical compound,
drug
doxycyclineSigmaCat# D9891
Chemical compound,
drug
DAPISigmaCat# 32670
Chemical compound,
drug
dimethylpidilate (DMP)Life TechnologiesCat# 21666
Chemical compound,
drug
5-bromo2’-deoxyuridineSigmaCat# B9285
Chemical compound,
drug
Denhardt’s Solution (50X)Life TechnologiesCat# 750018
Chemical compound,
drug
cycloheximideSigmaCat# C104450
Chemical compound,
drug
Ribonucleoside vanadyl
complexes (RVC)
New England BiolabsCat# S1402S
Chemical compound,
drug
Live-Dead Fixable AquaLife TechnologiesCat# L34957
Chemical compound,
drug
TO-PRO-3 IodideLife TechnologiesCat# T3605
Software, algorithmCLIP Toolkit (CTK)PMID:27797762
Software, algorithmSTAR AlignerPMID:23104886
Software, algorithmBowtie2PMID:22388286
Software, algorithmHOMERPMID:20513432
Software, algorithmGenomicRanges
(R Bioconductor)
PMID:23950696
Software, algorithmTxDb.Mmusculus.
UCSC.mm10.ensGene
(R Bioconductor)
DOI: 10.18129/B9.bioc.
TxDb.Mmusculus.UCSC.
mm10.knownGene
Software, algorithmTopGO (R Bioconductor)DOI: 10.18129/B9.bioc.
topGO
Adrian Alexa, Jorg Rahnenfuhrer
Software, algorithmedgeR (R Bioconductor)PMID:19910308
Software, algorithmHTseqPMID:25260700
Software, algorithmCluster 3.0PMID:14871861
Software, algorithmJava TreeviewPMID:15180930
Software, algorithmGene Set Enrichment
Analysis (GSEA)
PMID:16199517
Table 1
Oligonucleotide sequences
https://doi.org/10.7554/eLife.33057.022
Primer nameSequenceDescription
cloning
MJM9ATGACTCGAGGATCTCTCTGCCATCTACGAGAGCCmouse ZFP36 forward
XhoI primer
MJM10ATGAGCGGCCGCTCACTCAGAGACAGAGATACGATTGAAGATGGmouse ZFP36 reverse
NotI primer
MJM266ATGACTCGAGACCACCACCCTCGTGTCCmouse ZFP36L1 forward
XhoI primer
MJM267ATGAGCGGCCGCTTAGTCATCTGAGATGGAGAGTCTGC Gmouse ZFP36L1 reverse
NotI primer
MJM270ATGACTCGAGTCGACCACACTTCTGTCACCCmouse ZFP36L2 forward
XhoI primer
MJM271ATGAGCGGCCGCTCAGTCGTCGGAGATGGAGAGGmouse ZFP36L2 reverse
NotI primer
RT-qPCR
GP-fCATTCACCTGGACTTTGTCAGACTCLCMV RNA forward qPCR
GP-rGCAACTGCTGTGTTCCCGAAACLCMV RNA reverse qPCR
MJM432GATTGTGGGACATCCTGGTCmouse RPL10A forward
qPCR
MJM433TCAGACCCATGACTGCTGAGmouse RPL10A reverse
qPCR
MJM500AACATCGAAGACGGCTCTGTIFNG reporter forward
qPCR
MJM501GCGCTCTGTGTGGACAAGTAIFNG reporter reverse qPCR
MJM504CCACTACCTGAGCACCCAGTCD69 and TNF reporters
forward qPCR
MJM505GAACTCCAGCAGGACCATGTCD69 and TNF reporters
reverse qPCR
GAPDH-fGTCTCCTCTGACTTCAACAGCGhuman GAPDH forward
qPCR
GAPDH-rACCACCCTGTTGCTGTAGCCAAhuman GAPDH reverse
qPCR
HITS-CLIP and ribosome profiling
preA-L32/5rApp/GTGTCAGTCACTTCCAGCGG/3ddc/Pre-Adenylated 3' ligation
linker
RT1/5Phos/DDDCGATNNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
RT2/5Phos/DDDTAGCNNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
RT3/5Phos/DDDATCGNNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
RT4/5Phos/DDDGCTANNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
RT5/5Phos/DDDCTAGNNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
RT6/5Phos/DDDGATCNNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
RT7/5Phos/DDDAGTCNNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
RT8/5Phos/DDDTCGANNNNNNNAGATCGGAAGAGCGTCGT/iSp18/CACTCA/iSp18/CCGCTGGAAGTGACTGACIndexed RT primer
DP5-PEAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTforward PCR primer,
Illumina adapter
SP3-PECAAGCAGAAGACGGCATACGAGATCTCGGCATTCCTGCCGCTGGAAGTGACTGACACreverse PCR primer,
Illumina adapter
PE-R1ACACTCTTTCCCTACACGACGCTCTTCCGATCTsequencing primer
(standard Illumina read 1)

Additional files

Supplementary file 1

ZFP36 binding sites in CD4 +T cells 4 hr post-activation (attached spreadsheet).

Pan-ZFP36 HITS-CLIP peaks (A) in WT CD4 +T cells 4 hr post-activation (B) in Zfp36 KO CD4 +T cells 4 hr post-activation. (C) identified only in Zfp36 KO and not WT cells, and (D) pooled from WT and Zfp36 KO samples. (E) Cross-link induced truncation (CITS) cites from all pooled ZFP36 HITS-CLIP data (FDR < 0.01).

https://doi.org/10.7554/eLife.33057.023
Supplementary File 2

Gene Ontology enrichments for ZFP36 target mRNAs in CD4 +T cells, 4 hr post-activation (attached spreadsheet).

https://doi.org/10.7554/eLife.33057.024
Supplementary File 3

ZFP36 binding sites in CD4 +T cells 72 hr post-activation (attached spreadsheet).

https://doi.org/10.7554/eLife.33057.025
Transparent reporting form
https://doi.org/10.7554/eLife.33057.026

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  1. Michael J Moore
  2. Nathalie E Blachere
  3. John J Fak
  4. Christopher Y Park
  5. Kirsty Sawicka
  6. Salina Parveen
  7. Ilana Zucker-Scharff
  8. Bruno Moltedo
  9. Alexander Y Rudensky
  10. Robert B Darnell
(2018)
ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity
eLife 7:e33057.
https://doi.org/10.7554/eLife.33057