Temperature-sensitive eIF5A Mutant Accumulates Transcripts Targeted to the Nonsense-mediated Decay Pathway

Abstract

The highly conserved protein eIF5A found in Archaea and all eukaryotes uniquely contains the posttranslationally formed amino acid hypusine. Despite being essential the functions of this protein and its modification remain unclear. To gain more insight into these functions temperature-sensitive mutants of the human EIF5A1 were characterized in the yeast Saccharomyces cerevisiae. Expression of the point mutated form V81G in a ⌬eIF5A strain of yeast led to a strongly temperature-sensitive phenotype and to a significantly reduced protein level at restrictive temperature. The mutant showed accumulation of a subset of mRNAs that was also observed in nonsense-mediated decay (NMD)-deficient yeast strains. After short incubation at restrictive temperature the mutant exhibited increased half-lives of the intron containing CYH2 pre-mRNA and mature transcripts of NMD-dependent genes. Reduced telomere silencing and shortening was detected in the V81G mutant further supporting similarities to NMD-deficient strains. Our data suggest that eIF5A mediates important cellular processes like cell viability and senescence through its effects on the stability of certain mRNAs.The cellular physiology of mRNA processing, transport, localization, and turnover is central to the process of gene expression at the posttranscriptional level. Increasing evidence has been found for a close connection between mRNA degradation processes and the steps of translation. The highly conserved hypusine-containing protein eIF5A has been implicated in both of these aspects of RNA metabolism; however, its precise cellular function is not yet fully understood.Hypusine formation is a two-step enzymatic reaction catalyzed by deoxyhypusine synthase and then deoxyhypusine hydroxylase (1). The disruption of genes encoding either eIF5A or deoxyhypusine synthase in yeast leads to a lethal phenotype (2) demonstrating that the deoxyhypusine residue is essential for the function of eIF5A and thus for proliferation and cell survival. The genome of Saccharomyces cerevisiae contains two HYP genes (HYP1, alias TIF51B or ANB1, and HYP2, alias TIF51A) coding for eIF5A. These genes are differentially expressed under aerobic and anaerobic conditions (3) and share an identity of 90%. HYP genes of other higher eukaryotes, e.g. one of the two human HYP genes (encoding EIF5A1), can functionally replace these yeast-specific genes (4, 5).The eIF5A protein was first isolated from rabbit reticulocyte lysate ribosomes and classified as a translation initiation factor because in vitro the protein enhanced the building reaction of the first peptide bond measured as the yield of methioninepuromycin (6). However, cell fractionation revealed that only a small fraction of the protein associated with ribosomes (7). Moreover depletion or inactivation of the protein in S. cerevisiae reduced the global protein synthesis rate by only 30% (8, 9). Thus, the role of eIF5A as a translation initiation factor remains to be confirmed.eIF5A is an RNA-binding protein (10), and it was hypothesized to re...

Involvement of eukaryotic translation initiation factor 5A (eIF5A) in skeletal muscle stem cell differentiation

“…The GC7 treatment during the first two days of culture did not impair the process of cell differentiation possibly due to the low requirement of eIF5A during this period. The slight increase of the eIF5A transcript content during the whole period of GC7 treatment and the increase of MyoD1 transcript content in cells treated with GC7 for 48 h from the end of the second up to the end of the fourth day of differentiation may be associated to effects on mRNA stabilization as observed by others (Zuk and Jacobson, 1998;Schrader et al, 2006). However, the decline of the MyoD1 transcript content after prolongation of GC7 treatment revealed the possibility of extension of the initial phase of the differentiation program.…”

Revisiting an old acquaintance: role for eIF5A in diabetes

“…Supporting this notion, it has been reported that eIF5A itself has RNA-binding properties (18). Moreover, in the lower eukaryote S. cerevisiae, eIF5A appears to affect mRNA turnover/degradation (19,20), a process that is closely linked to nucleocytoplasmic mRNA transport. The present study by Maier and colleagues (3) identifies hypusine-modified eIF5A as a critical regulator of the nuclear export of Nos2 mRNA, further substantiating the notion that eIF5A has a nuclear function.…”

Posttranslational synthesis of hypusine: evolutionary progression and specificity of the hypusine modification

“…From differential display analysis of eIF5A-associated mRNAs, a number of mRNAs that are potential targets of eIF5A regulation have been reported (Xu et al, 2004). Genetic studies using S. cerevisiae harboring eIF5A temperature-sensitive mutants suggest a direct or indirect role of eIF5A in cell wall integrity, mRNA decay, actin polarization, cell cycle progression and anti-apoptotic protection (Zuk and Jacobson, 1998;Valentini et al, 2002;Zanelli and Valentini, 2005;Chatterjee et al, 2006;Schrader et al, 2006). Future studies will be directed toward identification of eIF5A binding partners (proteins and mRNA) and understanding how downstream effector molecules are involved in the expression of these pleiotropic phenotypes in the temperature-sensitive eIF5A mutant strains.…”