Study On Biological Function Of Various Truncations Of Human PRMT5 In E.coli And Direct Interaction With GM130

Arginine methylation is a prevalent post-translational modification found on both nuclear and cytoplasmic proteins.The methylation of arginine residues is catalyzed by the protein arginine Nmethyltransferase(PRMT) family of enzymes. Previously studies showed Arginine methylation participated in pre-mRNA processing, protein translation ,DNA transcription, signal transduction and DNA repair. Most PRMTs methylate glycine- and arginine-rich patches (GAR motifs) within their substrates. The complexity of the methylarginine mark is enhanced by the ability of this residue to be methylated in three different ways on the guanidino group: monomethylated (MMA), symmetrically dimethylated (sDMA) and asymmetrically dimethylated (aDMA), each of which has potentially different functional consequences.PRMTs are classified as type I, type II, type III or type IV enzymes. Type I and type II enzymes all catalyze the formation of an MMA intermediate, then type I PRMTs (PRMT1, 3, 4, 6 and 8)further catalyze the production of aDMA, whereas type II PRMTs (PRMT5, PRMT7 and FBXO11)catalyze the formation of sDMA. PRMT7 also exhibits type III enzymatic activity - the propensity to catalyze the formation of MMA on certain substrates and not proceed with sDMA catalysis. A type IV enzyme that catalyzes the monomethylation of the internal guanidino nitrogen atom has been described in yeast.Protein arginine methyltransferase 5 (PRMT5) has been implicated as an important regulator of many cellular processes and signaling pathways, including chromatin remodeling, RNA splicing, DNA transcription, and cell proliferation. Therefore, structural and functional studies on PRMT5 are quite important. The full length of PRMT5 gene was cloned into vector pGEX-4T-1, resulting in only low expression levels in Escherichia coli (E. coli). Here, we showed that the several N-terminal amino acids deletions could result in a significant increase in the amount of soluble fraction, while one of them did not affect the protein-arginine methyltransferase activity. And we also found that the N-terminal 15 amino acids region of PRMT5 may be important for the catalytic activity.The Golgi apparatus (GA) plays a central role in the post-translational modification, sorting, and transportation of proteins. Maintenance of GA structure and function depends on Golgin proteins and GRASPs (Golgi Re-Assembly Stacking Proteins).The posttranslational modification of golgins, such as phosphorylation of GM130, is critical for GA architecture2,4-9. The function of golgin methylation, however, has not been previously identified. Our group has shown that a PRMT5 complex contains several essential components involved in both GA stacking and vesicle tethering. One of them is GM130, a putative Golgi matrix protein.In order to show that GM130 directly interact with PRMT5, farwestern was applied. Our results show that the binding site for PRMT5 on GM130 maps to amino acids 180-619, a region conserved in the family. The same approach was used to show that the binding site for GM130 maps to the N-terminal 73 amino acids region.