The Functional Analysis Of Translation Initiation Factor EIF5B In A Rabidopsis

Eukaryotic protein synthesis requires numerous accessory factors that facilitate translation initiation,elongation,and termination.Translational control has been documented primarily at translation initiation,which involves multiple steps and protein factors.The eukaryotic translation initiation factor 5B(eIF5B)is a GTPase that acts late in the translation initiation process and is one of two universally conserved translation initiation factors(eIF1A and eIF5B).eIF5B interacts with the initiator tRNA,facilitates the final cleavage of the 20S pre-rRNA,and recruits the 60S ribosomal subunit to form the 80S ribosome in the final steps of translation initiation.However,the molecular mechanism by which eIF5B is involved in plant development and environmental response remains unknown.In this study,we analyze the function of eIF5B in Arabidopsis.The main results are as follows:1.We found that a missense mutation in an eIF5B gene in Arabidopsis(eIF5B-1;Atlg76810)prevents acclimation to high temperature.The thermotolerance-defective eIF5B-1 allele,a Ser substitution of a highly conserved Gly residue in Domain II,grows normally in the absence of stress.However,polysome profiling and RNA-seq analysis show delayed recovery of translation after heat stress and reduced polysome loading of certain heat shock protein(HSP)mRNAs,including ClpB3,ClpB4,CR88/HSP90.5,HSP17.6A and HSF7A.2.A second mutant allele that truncates eIF5B-1 by 30 aa,shows a significantly reduced growth rate.Polysome profiling reveals a dramatic increase in small polysomes and selective changes in polysome loading in the truncation mutant compared to wild type.RNA-seq data suggest that the mutant selectively decreases translation efficiency(TE)of ribosome-related,electron transport genes and auxin-regulated,and that many eIF5B-dependent mRNAs have short coding sequences.Thus,the phenotypes of these eIF5B-1 mutants appear to result from the requirement for optimal eIF5B activity for translation of specific mRNAs,indicating that even this universal translation factor has a significant role in post-transcriptional control of gene expression.3.Arabidopsis has three other eIF5B genes,none of which is capable of substituting for eIF5B-1 in thermotolerance or growth.One of these other genes,eIF5B-2(Atlg21160)shows differences in level and tissue distribution of expression explaining its inability to complement the eIF5B-1 mutants.The other two genes,eIF5B-3(Atlg76720)and eIF5B-4(Atlg76820)have clearly undergone evolutionary conversion to a non-functional state.The transcript of eIF5B-3 might be cleaved by ath-MIR2936,and the coding region of eIF5B-4 is deleted for a single"A" nucleotide,resulting in a premature stop codon.