Linking the 3′ Poly(A) Tail to the Subunit Joining Step of Translation Initiation: Relations of Pab1p, Eukaryotic Translation Initiation Factor 5B (Fun12p), and Ski2p-Slh1p

Abstract

The 3 poly(A) structure improves translation of a eukaryotic mRNA by 50-fold in vivo. This enhancement has been suggested to be due to an interaction of the poly(A) binding protein, Pab1p, with eukaryotic translation initiation factor 4G (eIF4G). However, we find that mutation of eIF4G eliminating its interaction with Pab1p does not diminish the preference for poly(A) ؉ mRNA in vivo, indicating another role for poly(A). We show that either the absence of Fun12p (eIF5B), or a defect in eIF5, proteins involved in 60S ribosomal subunit joining, specifically reduces the translation of poly(A) ؉ mRNA, suggesting that poly(A) may have a role in promoting the joining step. Deletion of two nonessential putative RNA helicases (genes SKI2 and SLH1) makes poly(A) dispensable for translation. However, in the absence of Fun12p, eliminating Ski2p and Slh1p shows little enhancement of expression of non-poly(A) mRNA. This suggests that Ski2p and Slh1p block translation of non-poly(A) mRNA by an effect on Fun12p, possibly by affecting 60S subunit joining.The 5Ј cap (7-methyl-GpppG. . .) and 3Ј poly(A) structures of eukaryotic mRNAs are both critically important for translation, for mRNA transport from the nucleus, and for mRNA stability in both the nucleus and the cytoplasm. The in vivo requirement for translation for the 3Ј poly(A) structure, estimated by electroporation of mRNAs into living cells, is about 50-fold, while that for the 5Ј cap is about 20-fold (11, 12).The effects of the 5Ј cap on translation are mediated by the cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E) (Cdc33p), which associates with eIF4G in the eIF4F complex to promote binding of the mRNA to the 40S ribosomal subunit (through eIF3). A 43S preinitiation complex consisting of the 40S subunit, eIF3, and eIF2-GTP-MettRNA iMet is thought to bind to an mRNA mediated by interaction between eIF3 and eIF4G. This complex scans from the cap at the 5Ј end of the mRNA to the first AUG. There, with the help of eIF5 (Tif5p) and eIF5B (Fun12p), 60S subunit joining occurs and translation begins (27; reviewed in references 9 and 37). 60S subunit joining requires both eIF5 and eIF5B. While eIF5 promotes GTP hydrolysis by eIF2, enabling release of the initiation factors from the 40S subunit, eIF5B has its own GTP binding activity and hydrolyzes GTP in a ribosome-dependent reaction (27).The role of the 3Ј poly(A) structure in mRNA translation is not yet completely clear. The poly(A) binding protein (Pab1p) is believed to mediate many of the effects of the 3Ј poly(A) structure (reviewed in reference 34). Because the poly(A) tail apparently has roles in nuclear processes as well as cytoplasmic events (22), dissecting these mechanisms is difficult. The PAB1 gene is essential, indicating that at least one of the functions of Pab1p is critical to the cell.

Free Poly(A) Stimulates Capped mRNA Translation in Vitro through the eIF4G-Poly(A)-binding Protein Interaction

“…Thus, the role of poly(A) in cappoly(A) synergy in vitro is to provide a scaffold for the binding of sufficient molecules of PABP to fully activate translation. However, these data do not rule out other possible roles for circularization in the context of an intact cell, such as reversal of Ski2p-mediated repression of nonpolyadenylated mRNA translation and enhanced 60 S subunit joining (30) or increased mRNA stability. The latter effect at least would be undetected in the depleted RRL system, in which polyadenylation and capping of a mRNA do not affect functional mRNA half-life (Ref.…”

Evidence that poly(A) binding protein has an evolutionarily conserved function in facilitating mRNA biogenesis and export

“…Both yeast (Tarun and Sachs 1996) and human (Imataka et al 1998) PABPs interact with the translation initiation factor eIF4G, thereby causing circularization of the mRNA via bridging its 5Ј and 3Ј termini (closed loop model [Jacobson 1996]). Such an interaction could facilitate formation of the 48S translation initiation complex de novo, the 60S ribosomal subunit joining, and/or translational reinitiation (Munroe and Jacobson 1990;Tarun and Sachs 1995;Kahvejian et al 2001;Searfoss et al 2001). However, the mechanism of translational stimulation may be more complex than just an interaction between PABP and eIF4G, as these two phenomena could be genetically uncoupled (Kessler and Sachs 1998).…”

Translation initiation by the c-myc mRNA internal ribosome entry sequence and the poly(A) tail

“…A bridging complex between the capbinding protein eIF4E, the adaptor protein eIF4G (I or II), and PABP bound to the 39 poly(A) tail can mediate circularization of the mRNA (Wells et al 1998). Biochemically, multiple mechanisms may contribute to enhance translation by: (1) promoting small ribosomal subunit recruitment via the 59-cap structure (Tarun and Sachs 1995;Kahvejian et al 2005); (2) stimulating 60S ribosomal subunit joining (Sachs and Davis 1989;Munroe and Jacobson 1990;Searfoss et al 2001); and/or (3) facilitating translation termination and ribosome recycling (Uchida et al 2002).…”

“…The poly(A) tail stimulates 48S initiation complex formation via the c-myc IRES Multiple mechanisms contribute to how poly(A) tails stimulate cap-dependent translation, including both 48S initiation complex formation and 60S ribosomal subunit joining (Sachs and Davis 1989;Munroe and Jacobson 1990;Tarun and Sachs 1995;Searfoss et al 2001;Kahvejian et al 2005). To determine how the poly(A) tail stimulates c-myc IRES-directed translation initiation, we analyzed its effect on 48S complex formation.…”