Fuctional Study Of Genes Responsible For M~7G Modified Nucleosides In TRNAs Of Arabidopsis Thaliana

The main function of transfer RNA(tRNA)is to bring amino acids into ribosomes,using the coding system between anticodon and codon on m RNA,to translate nucleotide sequences into protein sequences.tRNAs from all living organisms contain modified nucleosides,which are derivatives of the four canonical nucleosides including adenosine(A),uracil(U),guanosine(G)and cytosine(C).Modified nucleosides and nucleoside modification genes in bacteria and yeast have been studied extensively,but much less is known for higher plants.The most common type of tRNA modified nucleosides is methylation.m7G(N-7-methylguanosine)is a methylation of G often found at position 46 of tRNAs,this methylation need a protein complex consisting two subunits,Trm8 and Trm82 in yeast.This work focused on the identification of m7G modification genes in Arabidopsis thaliana,the main results are as the following:1.Based on protein sequence homology,we identified Arabidopsis candidate genes for m~7G methylation:At1g03110(At TRM82),At5g17660(At TRM8A)and At5g24840(At TRM8B);T-DNA mutant seeds were sreened to obtain homozygous knock out lines(#28 and#29)for At5g17660,old#12 for At1g03110;2.At5g17660 relative expression is reduced 95%in#28 homozygous mutant compared to wild type,whereas At1g03110 gene expression is reduced 32%in old#12 homozygous mutant.Nucleoside analysis suggested that#28 homozygous mutant had a 35%reduction on m~7G nucleoside level;3.Using tobacco transient transformation,we confirmed the subcellular protein localization of At5g17660-GFP to be both in the nucleus and cytoplasmic membranes;4.In vitro methylation of specific tRNA substrate suggested At5g17660 has methylation activity upon tRNA-Phe-GAA;yeast mutant complementation suggested that At5g17660 could rescue m~7G methylation in yeast trm8 mutant.In summary,At5g17660 is a TRM8 ortholog in Arabidopsis involved in m~7G nucleoside biosynthesis...