Translation profile of eIF5A dys1-1 hypusination mutant

Abstract

The recent advances in the study of eIF5A have shown a huge impact of its function in the protein synthesis process, more specifically, in the elongation step of mRNA translation. In the last years, eIF5A was involved in the translation of proteins containing sequences, or motifs, with three or more proline residues (poly-P) in a row, throughout the stimulation of the peptidyl transferase activity of the ribosome. It was shown that these poly-P sequences can act as ribosome stalling motifs and the presence of eIF5A between the P and E sites of the ribosome, with its hypusine residue (K51 in Saccharomyces cerevisiae; K50 in humans) localized into the Peptidyl Transferase Center (PTC), helps to relieve this stalling by facilitating protein bond formation. The same functional aspects were also observed for EF-P, the bacterial homolog of eIF5A. In fact, in the case of the bacterial homology, it was further shown that sequences upstream and downstream of the poly-P motifs can influence positively or negatively protein synthesis. Thus, it turned out to be important the identification of proteins that require eIF5A for their synthesis which may lead to a better comprehension the biological function of this translation factor and its influence on the G1/S cell cycle transition described in several organism. The experiments we are planning to perform will improve the current knowledge on the function of eIF5A, mainly in the control of gene expression at the translation level, by the use of translational profiling methodologies. In this scenario, we will analyze the translational profile (determination of mRNA pool associated to the ribosome), by RNA-seq technology, of the mutant dys1-1 (mutant of the first enzyme involved in hypusine formation, an essential posttranslational modification of eIF5A), known to impair the hypusination of eIF5A. As this mutant produces a non-functional eIF5A, we hope that the comparison between the results obtained with the wild type (Dys1) and the mutant (dys1-1) cells, using bioinformatics tools, will reveal the pool of mRNAs that are dependent on eIF5A for translation. Then, additional eIF5A mutants will also be included in this study. This activity plan is part of the Objective number 5 of the current thematic project (2010/50044-6) that aims to analyze the influence of eIF5A on the translational control of gene expression.