A single point mutation at the 3'-untranslated region of Ran mRNA leads to profound changes in lipopolysaccharide endotoxin-mediated responses.
By functional cDNA expression cloning, we have previously established that Ran is important in lipopolysaccharide (LPS) signaling. This was achieved by functional comparison between two cDNAs, differing by a single base substitution within the 3'-untranslated region of the cDNA. This point mutation results in a striking RNA conformational change. No dramatic difference in total RNA at steady state could be found between the two molecules. However, at the protein level, RanC/d (from 870C mRNA) was 5-10-fold higher than RanT/n (from 870T mRNA) and this difference was not observed in non-hematopoietic cells transduced with the same vectors. This tissue-specific difference correlated with a difference in LPS endotoxin responses in corresponding hematopoietic cells. Importantly, the amounts of Ran- C/d and RanT/n proteins were similar initially but the difference became obvious with time. Both Ran proteins migrated from the cytoplasm to the nucleus, but Ran from RanC/d migrated faster than that of RanT/n. RanT/n protein preferentially remained in the cytoplasm and its overall amount was reduced at steady state, consistent with its degradation by intracellular proteases known to be involved in LPS-mediated signal transduction. As the two proteins are identical, the faster RanC/d nuclear localization and a preferred initial cytoplasmic RanT/n distribution suggest a difference in mRNA intracellular localization between the two molecules, as dictated by their RNA structural difference. By pulse-chase experiments, RanC/d proteins are more resistant to degradation than RanT/n protein; there also appear to have two populations of RanT/n proteins, one may reside in the cytoplasm and the other, in the nucleus. More RanC/d GTPase accumulated in the nuclei would conceivably alter the potency of signal transduction and therefore down-modulate LPS-mediated biological responses.
Wong
P M
PM
Department of Pathology & Laboratory Medicine, Fels Institute, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA. Petermcw@aol.com
Yuan
Q
Q
Chen
H
H
Sultzer
B M
BM
Chung
S W
SW
D020413:3' Untranslated Regions / Q000737:chemistry / Q000235:genetics*; D000818:Animals; D001402:B-Lymphocytes / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D004731:Endotoxins / Q000633:toxicity; D006207:Half-Life; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000502:physiology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000633:toxicity*; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008809:Mice, Inbred C3H; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D021381:Protein Transport; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D012260:Ribonucleases / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D020413:3' Untranslated Regions; D004731:Endotoxins; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D012260:Ribonucleases; D020931:ran GTP-Binding Protein