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Research
Ching-Jin Chang
Academia Sinica
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7129-8092
License:
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Backgound: Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells and its mRNA destabilization activity is regulated by protein phosphorylation.
Methods: We generated an antibody against phospho-Serine 316 located at C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP in RAW264.7 cells using CRISPR/Cas9 gene editing was created to explore TTP functions.
Results: We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2), and dephosphorylated by Protein Phosphatase 2A (PP2A). Phosphorylation-mimic mutant of S316D resulted in dissociation with CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1.
Conclusions: Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation to regulate the TTP interaction with CCR4-NOT deadenylase complex.
Keywords
Tristetraprolin, phosphorylation, CNOT1, deadenylase
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CURRENT STATUS: Under Review
Version 1
Posted 04 Aug, 2020
No community comments so far
Editorial decision: Major revision
On 02 Feb, 2021
Review #2 received
Received 26 Jan, 2021
Review #1 received
Received 22 Jan, 2021
Reviewer #2 agreed
On 14 Jan, 2021
Reviewer #1 agreed
On 07 Jan, 2021
Reviewers invited
Invitations sent on 04 Sep, 2020
Editor assigned
On 31 Jul, 2020
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On 30 Jul, 2020
Editor invited
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Subject Areas
General Biochemistry
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This preprint is under consideration at Journal of Inflammation. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Ching-Jin Chang
Academia Sinica
Corresponding Author
ORCiD: https://orcid.org/0000-0001-7129-8092
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 04 Aug, 2020
No community comments so far
Editorial decision: Major revision
On 02 Feb, 2021
Review #2 received
Received 26 Jan, 2021
Review #1 received
Received 22 Jan, 2021
Reviewer #2 agreed
On 14 Jan, 2021
Reviewer #1 agreed
On 07 Jan, 2021
Reviewers invited
Invitations sent on 04 Sep, 2020
Editor assigned
On 31 Jul, 2020
First submitted
On 30 Jul, 2020
Submission checks complete
On 30 Jul, 2020
Editor invited
On 30 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Biochemistry
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Backgound: Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells and its mRNA destabilization activity is regulated by protein phosphorylation.
Methods: We generated an antibody against phospho-Serine 316 located at C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP in RAW264.7 cells using CRISPR/Cas9 gene editing was created to explore TTP functions.
Results: We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2), and dephosphorylated by Protein Phosphatase 2A (PP2A). Phosphorylation-mimic mutant of S316D resulted in dissociation with CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1.
Conclusions: Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation to regulate the TTP interaction with CCR4-NOT deadenylase complex.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
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