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Vol. 62, Issue 2, 423-431, August 2002

Inflammation Modulates the Interaction of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) I/Polypyrimidine Tract Binding Protein and hnRNP L with the 3'Untranslated Region of the Murine Inducible Nitric-Oxide Synthase mRNA

Malin Söderberg, Françoise Raffalli-Mathieu, and Matti A. Lang

Division of Biochemistry, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden

Interaction of two members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with the 3'untranslated region (UTR) of the murine inducible nitric-oxide synthase (iNOS) mRNA is demonstrated in this study. An iNOS RNA-protein complex is formed using protein extracts from untreated and septic shock treated mouse liver. UV cross-linking reveals that the complex consists of at least two proteins, with apparent molecular masses of 60 and 70 kDa, respectively. The 60-kDa protein binding site lies within a 112-nt pyrimidine-rich sequence, approximately 160 nt from the coding sequence, and the RNA-protein complex can be precipitated by a monoclonal antibody directed against hnRNP I [also named polypyrimidine tract binding protein (PTB)]. The 70-kDa protein binds a 43-nt sequence near the 3'end of the 3'UTR and is immunoprecipitated by a monoclonal antibody against hnRNP L. A computer-simulated conformation of the 3'UTR suggests that both binding sites reside in regions easily accessible for a protein. Supershifts of the native RNA-protein complex could only be achieved with anti-hnRNP L, suggesting that within this multiprotein RNA complex, only hnRNP L is exposed to the antibodies, whereas the hnRNP I/PTB is mainly responsible for its interaction with the mRNA. Up-regulation of iNOS by septic shock reduces the RNA-protein complex formation, thus showing that hnRNP I/PTB and hnRNP L binding to the iNOS mRNA is modulated by inflammation. This suggests a novel function for the two previously described proteins as regulators of the iNOS gene.

Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics


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