Translation repression via modulation of the cytoplasmic poly(A)-binding protein in the inflammatory response
- Mayo Clinic, United States
- University of Central Florida, United States
Figures
Figure 1 with 4 supplements
Global translation profiling of mouse primary BMDM-mediated inflammatory response.
(A) Workflow of parallel ribosome and RNA profiling during the BMDM-mediated inflammatory response. (B) Metagene plots show the rise and fall in 28-nt ribosome footprints (RFPs) density (reads per …
Figure 1—figure supplement 1
Reproducibility of Ribo-seq and GO analysis of differentially translated mRNAs in the inflammatory response.
(A) The ribosome profiling datasets in BMDM-mediated inflammatory response are highly reproducible. Scatterplots (lower left) show pairwise comparisons of the ribosome footprint (RFP) density …
Figure 1—figure supplement 2
TNF mRNA is translationally repressed during late stages of inflammatory response.
Reads from RNA-seq and Ribo-seq at each time point during the inflammatory response in the TNF locus were displayed.
Figure 1—figure supplement 3
Verification of translational repression of TNF mRNA at late time points in LPS-stimulated BMDMs.
(A) The apparent TEs of TNF mRNA in the BMDMs at 0, 1, 2, 4, 6 hr post LPS stimulation. The variation of the two biological replicates at 0 hr is likely due to low TNF mRNA expression at this time …
Figure 1—figure supplement 4
Zfp36 is a translational repressor.
The luciferase reporter was co-transfected into 293 T cells with either the lambdaN-Zfp36 expressing plasmid or the lambda-GFP expressing plasmid. The luciferase activity, the FLuc mRNA level, and …
Figure 2 with 1 supplement
Generation of a Zfp36 V5-epitope tag knock-in mouse for mechanistic studies on the endogenous Zfp36.
(A) Workflow for generating a Zfp36-V5 knock-in mouse via CRISPR/Cas9-mediated genome editing. (B) Genotyping of the Zfp36-V5 knock-in mice using the two primers shown in (A). (C) Specific and …
Figure 2—figure supplement 1
The Zfp36-V5 mouse is normal.
(A) The Zfp36-V5 mice were born in the predicted Mendelian ratio from heterozygous matings. The Zfp36-V5 mice from heterozygous matings were genotyped at 3–4 weeks old. (B) BMDMs from the Zfp36-V5 …
Figure 3 with 1 supplement
Endogenous Zfp36 interacts with Pabpc1 in LPS-stimulated BMDM.
(A) Workflow for the tandem mass tag (TMT) quantitative proteomics to identify the proteins associated with endogenous Zfp36 in LPS-stimulated BMDMs. (B) Zfp36-associated proteins identified by the …
Figure 3—figure supplement 1
The Zfp36-Pabpc1 interaction is independent of RNA.
A HA-Pabpc1 expressing plasmid was co-transfected into 293 T cells with a Zfp36-FLAG expressing plasmid. IP was performed using either an IgG control or an anti-FLAG antibody in the presence (+) or …
Figure 4 with 1 supplement
The Zfp36-Pabpc1 interaction is required for Zfp36-mediated translational repression.
(A) Luciferase reporters and Zfp36 and its truncations used in the tethering experiment. The blue box represents the λN polypeptide. (B) Luciferase activity, the FLuc mRNA, and translatability …
Figure 4—figure supplement 1
The Zfp36 N-terminus and C-terminus fragments do not interact with Pabpc1.
(A) Schematic presentation of Zfp36 full length, the Zfp36 N-terminus fragment, and the Zfp36 C-terminus fragment for the CoIP experiment. (B) The Zfp36 N-terminus and C-terminus fragments do not …
Figure 5
Transcriptome-wide identification of Zfp36 target mRNAs using CLIP-seq.
(A) A Table summarizing the number of uniquely mapped reads to the mouse genome for the CLIP-seq duplicates and RNA-seq. (B) Log plots of the number of uniquely mapped reads per gene from the …
Figure 6
The Zfp36-Pabpc1 interaction is important for Zfp36-mediated translational repression in LPS-stimulated BMDM.
(A) Expression of the Zfp36-mF fragment in LPS-stimulated BMDMs. BMDMs from the Zfp36-V5 mice were transduced with either an empty retroviral vector or a retroviral vector expressing the Zfp36-mF …
Figure 7
Zfp36 represses translation at similar steps as Pabpc1 regulates translation.
(A) Schematic presentation of the bicistronic luciferase reporter system for dissecting how Zfp36 represses translation. (B) Tethering Zfp36 does not change the mRNA levels of the bicistronic …
Additional files
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Supplementary file 1
TEs of mRNAs during the BMDM-mediated inflammatory response.
The first tab listed the TEs of the differentially translated mRNAs during the inflammatory response. The second tab listed the TEs of all the mRNAs during the inflammatory response.
- https://doi.org/10.7554/eLife.27786.017
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Supplementary file 2
Proteins identified by the TMT proteomic analysis.
- https://doi.org/10.7554/eLife.27786.018
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Supplementary file 3
Target mRNAs of the endogenous Zfp36 in activated BMDMs.
- https://doi.org/10.7554/eLife.27786.019
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Supplementary file 4
Plasmids, antibodies, chemicals, and oligoes used in this study.
- https://doi.org/10.7554/eLife.27786.020
