Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR.
We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3'-untranslated region (3'-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3'-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3'-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3'-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.
Linker
Katrin
K
Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Strasse 67, D-55101 Mainz, Germany.
Pautz
Andrea
A
Fechir
Marcel
M
Hubrich
Thomas
T
Greeve
Jobst
J
Kleinert
Hartmut
H
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism; D000954:Antigens, Surface / Q000378:metabolism*; D045744:Cell Line, Tumor; D016207:Cytokines / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000378:metabolism*; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology*; D015534:Trans-Activators / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D051816:Tristetraprolin
D020413:3' Untranslated Regions; D000954:Antigens, Surface; D016207:Cytokines; D004268:DNA-Binding Proteins; D051959:ELAV Proteins; D000067780:ELAV-Like Protein 1; C098770:ELAVL1 protein, human; D017874:Immediate-Early Proteins; C105789:KHSRP protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D015534:Trans-Activators; D051816:Tristetraprolin; C497552:ZFP36 protein, human; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II