Structure And Function Of The Tandem KH Domain Of MEX-3,the PUF Domain Of APUM5 And PRMT9

Living organisms are composed of an enormous number of biomacromolecules including proteins, DNAs, RNAs and other functional elements, which collaborate mainly through the interactions among them in all kinds of the regulating pathways for keeping all the mechanisms operating properly.In Caenorhabditis elegans, the KH-domain of MEX-3 protein mediates the activation or inhibition of the translation on messenger RNA (mRNA) according to its interaction with 3’-UTR of mRNA, so does the PUF-like protein, APUM5, in Arabidopsis thaliana. However, arginine methyltransferase PRMT9 in Danio rerio primarily methylates the arginines in the specific sites on some proteins to regulate its particular function, further interfering the downstream reaction process. Both the interaction between proteins and nucleotides or other proteins are presenting an orderly procedure for regulating the complement of cell activities. Intriguingly, the MEX-3 protein was checked in the P granule of Caenorhabditis elegans, containing KH domains in series and combining RNA to regulate the regeneration of totipotent stem cells and differentiation of cells during early embryogenesis. Moreover,PUF-like proteins are produced in many species. And APUM5 in Arabidopsis thaliana applies the PUF domain to recognize the genome of virus for opening the ego-defence mechanism. In addition, PRMT9 in Danio rerio takes part in the alternative splicing by methylating the splicing factor.In our work, we employed the Escherichia coli (E. coli) expression system to purify the KH domain of MEX-3 in Caenorhabditis elegans, PUF domain of APUM5 for Arabidopsis thaliana as well as the PRMT9 of Danio rerio by insect cell expression system. The proteins were purified by NTA affinity chromatography column, Hiload 16/60 Supdex 75/200, MonoS/Q and other highly efficient methods and then were detected the stability in the way of NMR and Dynamic light scattering experiments, after which we measured the interaction affinity using isothermal titration calorimetry (ITC) and accounted for the direct interaction of PRMT9 with the splicing factor SF3B2 according to pull-down experiments. X-ray structural analysis was applied to get the crystal of APUM5-RNA (the sequence of 3’-UTR in Tobacco mosaic virus (CMV)), which necessarily needs optimization for resolution.