title: Diphthamide modification on eukaryotic elongation factor 2 is needed to assure fidelity of mRNA translation and mouse development.
abstract: To study the role of the diphthamide modification on eukaryotic elongation factor 2 (eEF2), we generated an eEF2 Gly(717)Arg mutant mouse, in which the first step of diphthamide biosynthesis is prevented. Interestingly, the Gly(717)-to-Arg mutation partially compensates the eEF2 functional loss resulting from diphthamide deficiency, possibly because the added +1 charge compensates for the loss of the +1 charge on diphthamide. Therefore, in contrast to mouse embryonic fibroblasts (MEFs) from OVCA1(-/-) mice, eEF2(G717R/G717R) MEFs retain full activity in polypeptide elongation and have normal growth rates. Furthermore, eEF2(G717R/G717R) mice showed milder phenotypes than OVCA1(-/-) mice (which are 100% embryonic lethal) and a small fraction survived to adulthood without obvious abnormalities. Moreover, eEF2(G717R/G717R)/OVCA1(-/-) double mutant mice displayed the milder phenotypes of the eEF2(G717R/G717R) mice, suggesting that the embryonic lethality of OVCA1(-/-) mice is due to diphthamide deficiency. We confirmed that the diphthamide modification is essential for eEF2 to prevent -1 frameshifting during translation and show that the Gly(717)-to-Arg mutation cannot rescue this defect.
authors.affiliation: Microbial Pathogenesis Section, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA. shliu@niaid.nih.gov
authors.forename: Shihui
authors.initials: S
authors.lastname: Liu
authors.forename: Christopher
authors.initials: C
authors.lastname: Bachran
authors.forename: Pradeep
authors.initials: P
authors.lastname: Gupta
authors.forename: Sharmina
authors.initials: S
authors.lastname: Miller-Randolph
authors.forename: Hailun
authors.initials: H
authors.lastname: Wang
authors.forename: Devorah
authors.initials: D
authors.lastname: Crown
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Wein
authors.forename: Rajat
authors.initials: R
authors.lastname: Singh
authors.forename: Rasem
authors.initials: R
authors.lastname: Fattah
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Leppla
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000818:Animals; D001710:Biotin / Q000737:chemistry; D016466:CHO Cells; D006224:Cricetinae; D005347:Fibroblasts / Q000166:cytology; D017353:Gene Deletion; D006639:Histidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D015778:Minor Histocompatibility Antigens; D020652:Peptide Elongation Factor 2 / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D014176:Protein Biosynthesis; D025521:Tumor Suppressor Proteins / Q000235:genetics
chemical_list: C485679:Dph1 protein, mouse; D015778:Minor Histocompatibility Antigens; D020652:Peptide Elongation Factor 2; D010455:Peptides; D025521:Tumor Suppressor Proteins; D006639:Histidine; D000244:Adenosine Diphosphate; D001710:Biotin; C027527:diphthamide