An Archaeal Protein with Homology to the Eukaryotic Translation Initiation Factor 5A Shows Ribonucleolytic Activity

Abstract

To identify proteins that are involved in RNA degradation and processing in Halobacterium sp. NRC-1, we purified proteins with RNA-degrading activity by classical biochemical techniques. One of these proteins showed strong homology to the eukaryotic initiation factor 5A (eIF-5A) and was accordingly named archaeal initiation factor 5A (aIF-5A). Eukaryotic IF-5A is known to be involved in mRNA turnover and to bind RNA. Hypusination of eIF-5A is required for sequencespecific binding of RNA. This unique post-translational modification is restricted to Eukarya and Archaea. The exact function of eIF-5A in RNA turnover remained obscure. Here we show for the first time that aIF-5A from Halobacterium sp. NRC-1 exhibits RNA cleavage activity, preferentially cleaving adjacent to A nucleotides. Detectable RNA binding could be shown for aIF-5A purified from Halobacterium sp. NRC-1 but not from Escherichia coli, while both proteins possess RNA cleavage activity, indicating that hypusination of aIF-5A is required for RNA binding but not for its RNA cleavage activity. Furthermore, we show that the hypusinated form of eIF-5A also shows RNase activity while the unmodified protein does not. Charged amino acids in the N-terminal domain of aIF-5A as well as in the C-terminal domain, which is highly similar to the cold shock protein A (CspA), an RNA chaperone of E. coli, are important for RNA cleavage activity. Moreover our results reveal that activity of aIF-5A depends on its oligomeric state.

Cold adaptation of the Antarctic haloarchaea Halohasta litchfieldiae and Halorubrum lacusprofundi

“…litchfieldiae , several proteins associated with RNA degradation (Levy et al ., ; Hou et al ., ) also had higher abundance at low temperature: ribonuclease J (halTADL_2415) and the RNA‐binding DnaG protein (halTADL_0764). This extended to an archaeal homolog of eukaryotic initiation factor 5A (halTADL_2262) for which the precise function in RNA turnover is unclear, although the C‐terminal region is structurally similar to the RNA chaperone CspA (Wagner and Klug, ). For Hrr.…”

The critical role of RNA processing and degradation in the control of gene expression

“…It was shown that divalent cations are not required for their activity, and they were capable of cleaving yeast tRNA (Fusi et al, 1993;Shehi et al, 2001). A homologue of the eukaryotic initiation factor 5A (eIF-5A) called archaeal initiation factor 5A (aIF-5A), from Halobacterium salinarum, was also described as an RNase with activity in low salt concentrations without addition of MgCl 2 (Wagner & Klug, 2007). Furthermore, two different dehydrogenases were identified in the same organism, with RNase III-like properties and cleavage patterns dependent on MgCl 2 : an aspartate-semialdehyde dehydrogenase and acyl-CoA dehydrogenase (Evguenieva-Hackenberg et al, 2002).…”

Post-translation modification in Archaea: lessons fromHaloferax volcaniiand other haloarchaea

“…Hypusine is essential for activity of eIF5A/aIF5A (including that of Hbt. salinarum ), now considered important in translation elongation (Wagner and Klug, 2007; Saini et al, 2009; Park et al, 2010). In hypusine modification of eIF5A, deoxyhypusine synthase (DHS) transfers a 4-aminobutyl moiety from spermidine to the ε-amino group of the conserved lysine residue to form a dexoxyhypusine intermediate, which is hydroxylated to a hypusine residue by deoxyhypusine hydroxylase (DOHH) (Fig.…”

“…[Ne-(4-amino-2-hydroxybutyl)-L-lysine] is formed upon PTM of a conserved lysine residue and is found only in eukaryotic translation 'initiation' factor 5A (eIF5A) and the related archaeal aIF5A (Park et al, 1981(Park et al, , 2010Schumann & Klink, 1989;Bartig et al, 1992). Hypusine is essential for the activity of eIF5A/aIF5A (including that of H. salinarum), now considered important in translation elongation (Wagner & Klug, 2007;Saini et al, 2009;Park et al, 2010). In hypusine modification of eIF5A, deoxyhypusine synthase (DHS) transfers a 4-aminobutyl moiety from spermidine to the e-amino group of the conserved lysine residue to form a dexoxyhypusine intermediate, which is hydroxylated to a hypusine residue by deoxyhypusine hydroxylase (DOHH) (Fig.…”