The Study On The Association And The Functions Between Human P100Protein And G3BP Protein

Objectives:p100is a multiple functions Protein which is a co-factor of transcription and pre-mRNA splicing. It has4repeated SN domains and a Tudor-SN(TSN)domain. We has done a lot of research on p100recent years. We found that G3BP is one of the proteins pulled down by p100protein in our previous research. It is not known if the proteins interact in some specific situation or in common conditions, if they combine to each other directly or indirectly, or if the interaction needs some co-factors. Nor is it known which domain(s) mediates the interaction. So we carry out the following study on the mechanisms and functional consequences of the interaction between p100and G3BP proteins.Methods:Part Ⅰ:Interactions of p100and G3BP proteins in cells. First, we used the GST-p100fusion protein to pull down G3BP protein and coimmunoprecipitaion of G3BP and p100to confirm our previous observation. We also studied the colocalization of p100and G3BP by immunofluorescence microscopy.Part Ⅱ:Identification of interaction domains of p100and G3BP.1. We constructed several recombinant plasmids including5GST fusion constructs with different G3BP domains and one pcDNA3.1(+)-G3BP as template for in vitro translation.2. GST fusion proteins with various fragments of either G3BP or p100were used to pull down in vitro translated full length p100or G3BP labeled with35S to identify the interaction domains of p100and G3BP. Western Blot xperiments were also used to confirm the specific interactions of p100and G3BP.Part Ⅲ:Functional studies of the interactions between p100and G3BP in cells under stress. 1. We transfected pEGFP-CI-G3BP and pRFP-p100/p100-SN/p100-TSN plasmids into HeLa cells, then treated the cells with arsenite or heat shock and observed the colocalization of green and red fluorescence.2. Effect of mRNA metabolism by the association of p100and G3BP. We transfected COS7cells with pEGFP-G3BP or pSG5-p100or both and treated cells with arsenite or heat shock. Total RNA was extracted and used to study mRNA degradation.ResultsPart Ⅰ:We observed the partial colocalization of G3BP green fluorescence, which is mainly located in cytoplasm especially near the nucleus membrane, and p100red fluorescence, which spreads throughout the whole cell. Coimmunoprecipitation and Western Blot experiments also confirmed the interaction of both over-express and endogenous G3BP and p100.Part Ⅱ:We found that p100can bind to several domains, especially domain3and4of G3BP. The interaction between domain5of G3BP and p100was unstable, and probably unspecific. Domain2can not mediate the interaction. SN domain of p100mediates the interaction. The TSN and TD domains do not participate the interaction.Part Ⅲ:After arsenite or heat shock treatment, some green particles colocalized with the red ones. The number and size of particles increased with time. The SN domain, but not the TSN domain, of p100behaved the same way as the full length p100protein. We didn’t detect any difference in mRNA metabolism after transfection of various combination of plasmids after stress treatment.Conclusions:The interaction of G3BP and p100is direct and specific. The interacting domains are NTF2-like domain (domainl)、PxxP motif (domain3、RRM domain (domain4) of G3BP and SN domain of p100. G3BP is involved in stress granule formation and p100protein were recruited to the granules which is mediated by the SN domain.