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 b y functional comparison between two cDNAs, differing by a single base subst itution 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 R anT/n (from 870T mRNA) and this difference was not observed in non-hematopo ietic cells transduced with the same vectors. This tissue-specific differen ce correlated with a difference in LPS endotoxin responses in corresponding hematopoietic cells. Importantly, the amounts of RanC/d and RanT/n protein s 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 remain ed in the cytoplasm and its overall amount was reduced at steady state, con sistent with its degradation by intracellular proteases known to be involve d in LPS-mediated signal transduction. As the two proteins are identical, t he faster RanC/d nuclear localization and a preferred initial cytoplasmic R anT/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 degradati on 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. M ore RanC/d GTPase accumulated in the nuclei would conceivably alter the pot ency of signal transduction and therefore down-modulate LPS-mediated biolog ical responses.