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.

The 5′ cap (7-methyl-GpppG…) and 3′ 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 3′ poly(A) structure, estimated by electroporation of mRNAs into living cells, is about 50-fold, while that for the 5′ cap is about 20-fold (11, 12). The effects of the 5′ 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-Met-tRNA is thought to bind to an mRNA mediated by interaction between eIF3 and eIF4G. This complex scans from the cap at the 5′ 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 3′ 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 3′ 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. It has recently been observed that eIF4G and Pab1p bind to each other in the presence of poly(A) (38). This binding would be expected to circularize the mRNA, since eIF4G is attached to the 5′ cap by its association with eIF4E and to the 3′ poly(A) by its binding to Pab1p. It has been suggested that this is how the poly(A) tail functions to promote translation of mRNAs. However, mutations of eIF4G that eliminate the binding to Pab1p do not affect the growth rate of cells (40), suggesting Pab1p may activate translation by a different mechanism in vivo. Biochemical evidence suggested that the role of poly(A) was to promote joining of 60S subunits to the 40S subunit waiting at the initiator AUG (23), but these data showed only a twofold effect and doubts have been raised about this conclusion. A pab1ts mutant accumulates free 60S subunits at the nonpermissive temperature (32), just the result expected if 60S joining is the defective step. However, the relationship of the action of the joining factors Fun12p and Tif5p to the poly(A) structure has not been examined. Although there is a strict requirement for the 3′ poly(A) structure for translation, both in vivo and in vitro, recent work indicates that this requirement is one imposed by the inhibition of translation of non-poly(A) mRNAs by two homologous nonessential RNA helicases, Ski2p and Slh1p (20, 21, 35, 41). Mutations in SKI2 and SLH1, or other nonessential genes involved in this activity (SKI3, SKI7, and SKI8; see also reference 3), derepress translation of non-poly(A) mRNAs introduced by electroporation, the naturally poly(A)− mRNAs produced by the L-A and L-BC double-stranded RNA (dsRNA) viruses of yeast or the poly(A)− mRNAs produced from an RNA polymerase I promoter. Further, ski2Δ slh1Δ double mutants treat poly(A)+ and poly(A)− mRNAs the same, translating them at the same rate and for the same duration (35). Ski2p, Ski3p, and Ski8p are found in a cytoplasmic complex (4), but their precise role is unclear. As in yeast, there are two human Ski2p homologues (8, 19, 24, 30). Here we sought to relate the effects of Ski2p and Slh1p in blocking the translation of non-poly(A) mRNA to the translation-promoting effects of Fun12p and Pab1p. Our results support a role of the poly(A) structure in 60S ribosomal subunit joining promoted by Fun12p (eIF5B) and Tif5p (eIF5) but argue against a role for the Pab1p-eIF4G interaction in mediating the poly(A) requirement for translation. We find that the derepression of translation of non-poly(A) mRNA by the ski2Δ slh1Δ double mutation is abrogated by deficiency of Fun12p, suggesting a model in which Ski2p and Slh1p inhibit Fun12p action on mRNAs lacking a 3′ poly(A) structure.