A Single Point Mutation at the 3′-Untranslated Region of Ran mRNA Leads to Profound Changes in Lipopolysaccharide Endotoxin-mediated Responses

Quan Yuan,Hong Chen,Barnet M Sultzer,Siu-Wah Chung,Peter M.C Wong

doi:10.1074/jbc.m105400200

Abstract

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.

Highlights

Using a functional cDNA expression cloning strategy, Kang et al [23] isolated a cDNA whose expression in C3H/HeJ splenic B cells confers the ability to respond to LPS
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
The data suggest the presence of two types of proteins in RanT/n-transduced cells: one gets degraded more rapidly than the other, whereas the curve for Ran in RanC/d-transduced cells appears to be a single species. Results from both computer analyses as well as gel retardation assay reveal a significance structural difference that exists between the RanT/n and RanC/d RNA molecules. This difference co-localizes with that of the point mutation at position 870 in the 3Ј-UTR of the gene, and in regions distal to this point mutation no significant structural difference is observed (Fig. 1)

Summary

Introduction

Using a functional cDNA expression cloning strategy, Kang et al [23] isolated a cDNA whose expression in C3H/HeJ splenic B cells confers the ability to respond to LPS. The assay employed in that study was one of the functional B cell mitogenic assays originally used to define the defect in C3H/HeJ mice and the genetic basis for LPS responses [1, 2] This gene, Ran, encodes for a small G protein, Ran GTPase, which has been shown to be involved in diverse biological functions. RanT/n mRNA produces the same amount of protein initially as the RanC/d mRNA, but with time the overall level of RanT/n protein was reduced at steady state compared with RanC/d protein, which is a function of their differential intracellular localization These differences correlated with the ability of the transduced macrophages to production proinflammatory cytokine TNF␣ in their response to endotoxin LPS stimulation

Methods

Results

Conclusion