Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.
Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.
Kahvejian
Avak
A
Department of Biochemistry, McGill Cancer Center, McGill University, Montreal, Quebec, H3G 1Y6,Canada.
Svitkin
Yuri V
YV
Sukarieh
Rami
R
M'Boutchou
Marie-Noël
MN
Sonenberg
Nahum
N
D000818:Animals; D002457:Cell Extracts; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D039642:Eukaryotic Initiation Factors / Q000502:physiology*; D006801:Humans; D039122:Poly(A)-Binding Protein I / Q000502:physiology*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism
D002457:Cell Extracts; D039603:Eukaryotic Initiation Factor-4G; D039642:Eukaryotic Initiation Factors; D039122:Poly(A)-Binding Protein I