title: An efficient system for cap- and poly(A)-dependent translation in vitro.
abstract: The 3' poly(A) tail of eukaryotic messenger RNAs (mRNAs) acts synergistically with the 5' cap structure to enhance translation. This phenomenon has been explained by the simultaneous binding of poly(A)-binding protein (PABP) and a cap-binding protein (eIF4E) to eIF4G that results in the circularization of the mRNA (closed-loop model). We developed a robust cell-free protein synthesis system to study poly(A)-dependent translation. In nuclease-treated extracts of Krebs-2 ascites cells, the mRNA poly(A) tail and the cap structure synergistically stimulate translation. We also describe an efficient procedure for depleting PABP from translation extracts. Greater than 98% of PABP can be depleted from extracts by preincubation with either of the PABP-interacting proteins (Paip2 or Paip1) coupled to beads, and these depleted extracts fail to support efficient translation of poly(A)+ mRNAs. Translation activity is restored to depleted extracts by the addition of recombinant PABP.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
authors.forename: Yuri V
authors.initials: YV
authors.lastname: Svitkin
authors.forename: Nahum
authors.initials: N
authors.lastname: Sonenberg
mesh_terms: D000818:Animals; D001201:Ascites / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D002474:Cell-Free System; D002846:Chromatography, Affinity / Q000379:methods*; D039561:Eukaryotic Initiation Factor-4E / Q000235:genetics* / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008807:Mice, Inbred BALB C; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D012315:RNA Caps; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D002352:Carrier Proteins; D039561:Eukaryotic Initiation Factor-4E; C111580:PAIP1 protein, human; D010448:Peptide Initiation Factors; D012315:RNA Caps; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins