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.
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.
Söderberg
Malin
M
Division of Biochemistry, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden. malin.hobro-soderberg@farmbio.uu.se
Raffalli-Mathieu
Françoise
F
Lang
Matti A
MA
D020413:3' Untranslated Regions / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007249:Inflammation / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D008970:Molecular Weight; D046912:Multiprotein Complexes; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D009690:Nucleic Acid Conformation; D038941:Polypyrimidine Tract-Binding Protein; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000378:metabolism*; D012772:Shock, Septic / Q000378:metabolism
D020413:3' Untranslated Regions; D034664:Heterogeneous-Nuclear Ribonucleoprotein L; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D038941:Polypyrimidine Tract-Binding Protein; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse