eIF5A Promotes Translation of Polyproline Motifs

Abstract

SUMMARY Translation factor eIF5A, containing the unique amino acid hypusine, was originally shown to stimulate methionyl-puromycin synthesis, a model assay for peptide bond formation. More recently, eIF5A was shown to promote translation elongation; however, its precise requirement in protein synthesis has remained elusive. Here we use in vivo assays in yeast and in vitro reconstituted translation assays to reveal a specific requirement for eIF5A to promote peptide-bond formation between consecutive proline residues. Addition of eIF5A relieves ribosomal stalling during translation of three consecutive proline residues in vitro, and loss of eIF5A function impairs translation of polyproline-containing proteins in vivo. Hydroxyl radical probing experiments localized eIF5A near the E site of the ribosome with its hypusine residue adjacent to the acceptor stem of the P-site tRNA. Thus, eIF5A, like its bacterial ortholog EFP, is proposed to stimulate the peptidyl-transferase activity of the ribosome and facilitate the reactivity of poor substrates like proline.

Biological Relevance and Therapeutic Potential of the Hypusine Modification System

“…The highly specific and conserved post-translational hypusine modification system has a crucial function in translation regulation (9,11), and aberrant activation or inhibition is implicated in disparate disorders like cancer and infectious diseases. Notably, hypusine modification of eIF-5A displays an attractive platform for therapeutic intervention.…”

“…eIF-5A Targets Are Organized in Highly Connected ProteinProtein Interaction Networks-It was recently shown that fully hypusinated eIF-5A1 is a sequence-specific elongation factor, regulating the translation of proteins containing consecutive prolines in the form of repetitive PPP (Pro-Pro-Pro) and/or PPG (Pro-Pro-Gly) sequence units (11). Translation of proteins working together in complexes is regulated in a "proportional synthesis strategy" to provide the cell with the correct stoichiometric amounts for each member of the protein complex (53).…”

“…Hypusine is mandatory for eIF-5A activity and function (7,8), and only hypusinated eIF-5A was shown to control translation elongation as well as the nuclear export of spe-cific mRNAs (9,10). Moreover, recent studies linked eIF-5A to the translational control of proteins containing consecutive proline residues (11).…”

Using mitoribosomal profiling to investigate human mitochondrial translation

“…There are many features that can contribute to the pausing and processivity of the synthetic machinery as it translates its template mRNA and it is becoming apparent that this is not as simple as, or restricted to, the secondary structure of the RNA ahead of the ribosome. Pausing may also be regulated by the abundance of charged tRNA, the number of consecutive codons for the same amino acid 2 , the charge of the newly synthesised peptide, folding and secondary structure of the peptide fragment within the exit tunnel 3, 4 , the influence of non-canonical wobble bases in codon:anticodon pairing 5 or by chemical damage of mRNAs or their degradation such that they lack a stop codon. Transient accumulation of ribosomes can also be observed at sites of translation initiation, whilst ribosomes are localising to particular subcellular locations 6 or during recruitment of chaperones or regulatory factors 7 .…”

Distinct XPPX sequence motifs induce ribosome stalling, which is rescued by the translation elongation factor EF-P

“…EF-P homologs exist in all archaea and eukaryotes, termed aIF-5A and eIF-5A, respectively (11). Yeast eIF-5A has recently been shown to also rescue translational stalling at polyprolinestretches (12). Like EF-P, a/eIF-5A is also posttranslationally modified, but via hypusinylation rather than lysinylation (11).…”