Comparison of the capacity of different viral internal ribosome entry segments to direct translation initiation in poly(A)-dependent reticulocyte lysates.
Polyadenylation stimulates translation of capped eukaryotic mRNAs and those carrying picornaviral internal ribosome entry segments (IRESes) in vivo. Rabbit reticulocyte lysates (RRL) reproduce poly(A)-mediated translation stimulation in vitro after partial depletion of ribosomes and ribosome-associated factors. Here, we have evaluated the effects of varying different parameters (extent of extract depletion, cleavage of eIF4G, concentrations of KCl, MgCl(2) and programming mRNA) on IRES-driven translation efficiency and poly(A)-dependency in ribosome-depleted RRL. For comparison, the study included a standard capped, polyadenylated mRNA. Dramatic differences were observed in the abilities of the different IRESes to direct translation in ribosome-depleted extracts. While the hepatitis A virus IRES was incapable of driving translation in physiological conditions in depleted RRL, mRNAs carrying the foot-and-mouth disease virus and hepatitis C virus IRESes were translated significantly better than a standard cellular mRNA in the same conditions. Indeed, the capacities of these IRESes to direct translation in ribosome-depleted RRL were similar to those reported previously in certain cell lines. Both the abilities of the IRESes to drive translation and their individual salt optima in ribosome-depleted extracts suggest that these elements have dramatically different affinities for some component(s) of the canonical translation machinery. Finally, using poliovirus as an example, we show that the ribosome-depleted system is well suited to the study of the translational capacity of naturally occurring IRES variants.
Paulous
Sylvie
S
Unité de Régulation de la Traduction Eucaryote et Virale, CNRS URA 1966, Institut Pasteur, 25 rue du Dr. Roux, 75724 Paris Cedex 15, France.
Malnou
Cécile E
CE
Michel
Yanne M
YM
Kean
Katherine M
KM
Borman
Andrew M
AM
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002474:Cell-Free System; D010450:Endopeptidases / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D011061:Poly A / Q000235:genetics*; D011189:Potassium Chloride / Q000494:pharmacology; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014780:Viruses / Q000235:genetics*
D039603:Eukaryotic Initiation Factor-4G; D012333:RNA, Messenger; D015636:Magnesium Chloride; D011061:Poly A; D011189:Potassium Chloride; D010450:Endopeptidases