The control of mRNA deadenylation by MAPKAP kinase 2 and tristetraprolin
Author(s)
Marchese, Francesco Paolo
Type
Thesis
Abstract
The abundance of a given mRNA is a function of its rates of synthesis and
degradation. The regulation of mRNA stability is therefore an important means
of modulating gene expression. Control of mRNA stability is mediated by cisacting
sequences within 5’ or 3’ untranslated regions (UTR), such as
adenosine/uridine-rich elements (ARE) contained in the 3’ UTRs of many
inflammatory mediator mRNAs, and by the action of trans-acting RNA-binding
factors that interact with these cis-acting elements. Tristetraprolin (TTP) is an
ARE-binding protein that destabilises inflammatory mRNAs by promoting their
deadenylation. TTP is phosphorylated and inactivated in the cell by MAPKactivated
protein kinase (MK)-2, a kinase downstream of p38, which is
activated by pro-inflammatory stimuli. I have successfully set up an in vitro
assay to investigate TTP-directed deadenylation of ARE-containing mRNAs
(IVDA, in vitro deadenylation assay) and this assay was used to investigate
different aspects of the deadenylation mechanism. I show that a TTP-directed
deadenylase activity is present in HeLa and RAW 264.7 cells and that the
mechanism whereby TTP promotes deadenylation of ARE-containing RNA is
regulated by MK2 in vitro. In fact, phosphorylation of Ser-52 and Ser-178 of
TTP by MK2 resulted in inhibition of TTP-directed deadenylation of AREcontaining
RNA. The use of 14-3-3 protein antagonists showed that regulation
of TTP-directed deadenylation by MK2 is independent of 14-3-3 binding to
TTP. To investigate the mechanism whereby TTP promotes deadenylation, it
was necessary to identify the deadenylases involved. The carbon catabolite
repressor protein (CCR)4-CCR4-associated factor (CAF)1 complex was
identified as the major source of deadenylase activity in HeLa cells responsible
for TTP-directed deadenylation. CAF1a and CAF1b were found to interact with
TTP in an RNA-independent fashion. MK2 phosphorylation was found to
reduce the ability of TTP to promote deadenylation by inhibiting the recruitment
of CAF1 deadenylase in a mechanism that does not involve sequestration of
TTP by 14-3-3.
degradation. The regulation of mRNA stability is therefore an important means
of modulating gene expression. Control of mRNA stability is mediated by cisacting
sequences within 5’ or 3’ untranslated regions (UTR), such as
adenosine/uridine-rich elements (ARE) contained in the 3’ UTRs of many
inflammatory mediator mRNAs, and by the action of trans-acting RNA-binding
factors that interact with these cis-acting elements. Tristetraprolin (TTP) is an
ARE-binding protein that destabilises inflammatory mRNAs by promoting their
deadenylation. TTP is phosphorylated and inactivated in the cell by MAPKactivated
protein kinase (MK)-2, a kinase downstream of p38, which is
activated by pro-inflammatory stimuli. I have successfully set up an in vitro
assay to investigate TTP-directed deadenylation of ARE-containing mRNAs
(IVDA, in vitro deadenylation assay) and this assay was used to investigate
different aspects of the deadenylation mechanism. I show that a TTP-directed
deadenylase activity is present in HeLa and RAW 264.7 cells and that the
mechanism whereby TTP promotes deadenylation of ARE-containing RNA is
regulated by MK2 in vitro. In fact, phosphorylation of Ser-52 and Ser-178 of
TTP by MK2 resulted in inhibition of TTP-directed deadenylation of AREcontaining
RNA. The use of 14-3-3 protein antagonists showed that regulation
of TTP-directed deadenylation by MK2 is independent of 14-3-3 binding to
TTP. To investigate the mechanism whereby TTP promotes deadenylation, it
was necessary to identify the deadenylases involved. The carbon catabolite
repressor protein (CCR)4-CCR4-associated factor (CAF)1 complex was
identified as the major source of deadenylase activity in HeLa cells responsible
for TTP-directed deadenylation. CAF1a and CAF1b were found to interact with
TTP in an RNA-independent fashion. MK2 phosphorylation was found to
reduce the ability of TTP to promote deadenylation by inhibiting the recruitment
of CAF1 deadenylase in a mechanism that does not involve sequestration of
TTP by 14-3-3.
Date Issued
2011-01
Date Awarded
2011-06
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)
Advisor
Dean, Jonathan
Saklatvala, Jeremy
Publisher Department
Kennedy Institute of Rheumatology
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)