Abstract
ABSTRACTMCPIP1 and IER3 are recently described proteins essential for maintenance of immune homeostasis. IER3 is involved in the regulation of apoptosis and differentiation and has been shown lately to protect activated T cells and macrophages from apoptosis. MCPIP1 is an RNase critical for controlling inflammation-related mRNAs. MCPIP1 interacts with and degrades a set of stem-loop-containing mRNAs (including IL-6). Our results demonstrate the involvement of MCPIP1 in the regulation of IER3 mRNA levels. A dual luciferase assay revealed that over-expression of MCPIP1 resulted in a decrease of luciferase activity in the samples co-transfected with constructs containing luciferase CDS attached to IER3 3′UTR. We identified a stem-loop structure similar to that described to be important for destabilization of the IL-6 mRNA by MCPIP1. Examination of IER3 3′UTR sequence, structure and evolutionary conservation revealed that the identified stem-loop is buried within a bigger element. Deletion of this fragment abolished the regulation of IER3 3′UTR-containing transcript by MCPIP1. Finally, using immunofluorescence-combined single-molecule RNA FISH we have shown that the MCPIP1 protein co-localizes with IER3 mRNA. By this method we also proved that the presence of the wild-type NYN/PIN-like domain of MCPIP1 correlated with the decreased level of IER3 mRNA. RNA immunoprecipitation further confirmed the interaction of MCPIP1 with IER3 transcripts in vivo.
Highlights
Inflammation is a crucial component of the immune response that allows organisms to deal with the loss of cellular and tissue homeostasis, repair tissue damage and fight invading pathogens
Immediate early response gene 3 (IER3) 3′untranslated regions (UTRs) contains conserved elements potentially recognized by MCPIP1 We demonstrated previously that transcription factor Elk-1 is involved in IL-1β-dependent regulation of ZC3H12A
MCPIP1 protein interacts with IER3 mRNA in vivo By the use of smRNA FISH we have proved co-localization of MCPIP1 with IER3 transcript, confirmation of their direct interaction required employment of another method
Summary
Inflammation is a crucial component of the immune response that allows organisms to deal with the loss of cellular and tissue homeostasis, repair tissue damage and fight invading pathogens. Exaggerated response may lead to severe tissue damage. For a rapid induction and efficient resolution of the inflammation, gene expression in cells of the immune system is tightly regulated at the transcriptional and post-transcriptional level (Anderson, 2008, 2009, 2010; Hao and Baltimore, 2009; Medzhitov and Horng, 2009). Immediate early response gene 3 (IER3) belongs to the group of genes rapidly activated during inflammation. Its expression is induced by pro-inflammatory cytokines such as TNF and IL-1β and Department of Cell Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Cracow 30-387, Poland
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