Screening Of Proteins Interacting With Prmt5 By Yeast Two-Hybrid System In Medaka,Oryzias Latipes

Prmt5,a type ? protein arginine methyltransferase,plays important roles in regulation of gene expression,RNA processing,cell growth and differentiation,and is also essential for signal transduction and germ cell development.We screened the proteins interacting with Prmt5 of medaka by yeast two-hybrid technology and studied the interaction domains preliminarily.Firstly,medaka prmt5 of 1905 bp was isolated and subcloned into the bait vector as pGBKT7-prmt5 The reporter genes were not activated and the fusion proteins were not toxic to the cells after transforming yeast strains,AH 109 and Y187,with pGBKT7-prmt5.Secondly,the yeast two-hybrid cDNA library was constructed by SMARTTM cDNA library construction kit with strain AH 109.The seperated cDNA was dispersive in the range of 100?3000 bp.The cDNA library was about 1.4×106 cfu,the inserts of cDNA were 0.5?3.0 kb,about 1 kb in average,and the recombination rate of the colonies was 100%.The results indicated the diversity and length of cDNA fragments of the library were suitable for screening.Thirdly,the positive colonies were identified by mating Y187 cells containing pGBKT7-prmt5 and AH109 cells containing cDNA library on plates with SD/-His-Leu-Trp-Ade dificient medium and X-?-gal.Eight proteins were confirmed interacting with Prmt5 from 69 preliminary positive clonies after four passages,X-?-gal analysis and backcross test.The proteins interacting with Prmt5 are methylosome protein 50-like(Mep50),apolipoprotein A-I-like,transcript variant X2,PR domain zinc finger protein 1-like(Prdmla),sialic acid-binding Ig-like lectin 12-like(Siglecl2),PR domain zinc finger protein 1-like(Prdmlb),multifunctional protein ADE2-like,NADH dehydrogenase subunit 4 and sciellin.Finally,a weak interaction of Prmt5 with Prdm1a/Prdm1b and a strong interaction of Mep50 with Prdm1a/Prdm1b were observed by yeast two-hybridization.The colony sizes were not obviously different in the truncated and mutated sequences of mep50 with Prdm1a/Prdm1b.The interaction domain between Prdm1a/Prdm1b and Prmt5 or Mep50 need further studies by yeast two-hybrid and co-immunoprecipitation.This study is the first about screening the proteins interacting with Prmt5 in fish and will promote the study of the roles and mechanisms of Prmt5 in fish.