Functional Study Of Arabidopsis TRNA-m~1G37 Methyltransferase AtTrm5b

The tRNA-m¹G37 modification exists among eukaryotes,bacteria and archaea conservatively.In eukaryotes,the methyltransferase,catalyzing the m¹G37modification,is responsible for Trm5.It has been shown that lack of m¹G37 in yeast and humans severely affects cell growth and negatively impacts multiple life activities.In Arabidopsis,At Trm5a was identified as a tRNA-m¹G37methyltransferase catalyzing the onset of m¹G modification at 37th site of the Arabidopsis nucleus tRNA.In attrm5a deletion mutants,negative phenotypes,including poor growth and shortened filaments,were observed,along with altered ribosome content.AtTrm5b,a homologous protein of At Trm5a,showed a purely syngeneic lethal phenotype in its mutants.Based on the above results,this study investigated the function of AtTrm5b as a tRNA-m¹G37 methyltransferase of Arabidopsis thaliana and the possible reasons for the creation of pure lethality through multifaceted analysis,and the results obtained in this paper are as follows.1.Bioinformatics analysis showed that the NLPK conserved motif exists in the AtTrm5b coding region methyltransferase Domain,which is a class I methyltransferase conserved motif.AtTrm5b has high similarity to the D2 and D3structural domains of At Trm5a protein,but AtTrm5b contains a longer N-terminal sequence.2.Subcellular localization:AtTrm5b protein is localized in mitochondria,and truncation of the predicted mitochondrial targeting guide sequence does not affect its localization.3.In vitro methylation:AtTrm5b recognizes Arabidopsis endogenous mitochondrial tRNA_UGG^Proas a substrate and catalyzes the synthesis of m¹G37.And with the increase of,AtTrm5b protein concentration,the abundance of m¹G37 modification was significantly enhanced.4.Yeast heterologous complementation:in brewer’s yeast trm5 mutant lacking m¹G37 and having a growth retarded phenotype,its growth phenotype was restored with the introduction of AtTrm5b,while the m¹G modification was back-complemented in the complementary material.5.Purely syngeneic lethal rescue:Using the promoter-driven,AtTrm5b expression of the early embryo-specific expression gene LEC1,the attrm5b heterozygous mutant material was back-complemented.By heritability identification and phenotypic observation,the embryonic expression of attrm5b caused effects on germination rate and root length,and other phenotypes are to be further investigated.The above data demonstrate that AtTrm5b,as a methyltransferase gene of m¹G37,and At Trm5a may have different functions and genetic significance.