Mechanisms and regulation of translation initiation

a. Preinitiation complex assembly

We have made progress in elucidating the in vivo molecular functions of eIFs in PIC assembly and identifying critical residues in each factor involved in different steps of the process. These advances were aided by a technique we developed for crosslinking eIFs, Met-tRNA_i^Met and mRNAs to 40S subunits in living cells to preserve native PICs during fractionation by sedimentation through sucrose gradients. Combining this assay with genetic analysis of GCN4 expression and biochemical characterization of eIF interactions in vivo and in vitro, we provided several lines of evidence that assembly of eIFs 1, 2, 5, and 3 into the MFC enhances 43S PIC assembly in vivo. First, we identified mutations in specific residues of the eIF3c/NIP1 NTT—the segment linking c/NIP1 to the eIF5-CTT, eIF1, and (indirectly) eIF2b—that weaken these interactions in vitro, reduce eIF2 and eIF5 occupancy of native PICs, and confer a Gcd^- phenotype suppressible by hc-TC ⁵ (Fig. 3). Second, we showed that depleting subunits of eIF2 or eIF3 using conditional “degron” alleles, substantially decreases 40S occupancies of all MFC components in native PICs, showing that MFC is rate-enhancing (but not essential) for assembly or stability of PICs in vivo ⁸. Third, we identified point mutations in surface-exposed residues 93-97 of eIF1 that confer a Gcd^- phenotype and impair MFC stability and 40S recruitment of MFC components in vivo (Fig. 3).

Analysis of eIF1 mutations was greatly enhanced by our collaboration with Jon Lorsch and his colleagues, who measured rate constants and equilibrium binding constants for specific reactions in PIC assembly. This allowed us to show that the Gcd^- mutations 9,12 (in the unstructured NTT) and G107R (in the globular domain) lower the rate of TC loading on 40S subunits without decreasing 40S binding of eIF1 itself ⁹ (Fig. 3). We made the same finding for Gcd^- point mutations (F131,F131), or complete truncation (DC), of the unstructured CTT of eIF1A ^10,11 (Fig. 4).

Interestingly, the 131,133 mutation in eIF1A eliminates the ability of eIF1 to promote TC loading in the reconstituted system, indicating a functional interaction between eIF1 and the eIF1A CTT. A Gcd^- point mutation in the OB-fold of eIF1A (66-70) was found to reduce TC loading indirectly by decreasing 40S binding of eIF1A itself, supporting the idea that eIF1A binds in the 40S A-site in a manner similar to that of related, bacterial translation factor IF1 ¹² (Fig. 4). The Gcd^- eIF1A mutations reduce the levels of MFC components in native PICs, further demonstrating the interdependency in eIF binding to 40S subunits in vivo ^10,11.

We previously showed that recruitment of eIF3 to 40S subunits in vivo is promoted by N- and C-terminal segments of a/TIF32 and c/NIP1 ¹³. Recently, we implicated the RRM domain in the N-terminus of b/PRT1 in eIF3 recruitment to 40S subunits. Mutating the RNP1 motif (rnp1) weakens PRT1 binding to a/TIF32 and j/HCR1 and lowers 40S binding of all eIF3 subunits except j/HCR1. Overexpressing HCR1 suppresses the Ts^- phenotype and 40S binding defect of other eIF3 subunits in rnp1 cells, while deleting HCR1weakens 40S occupancy of eIF3 in vivo. These results show that the j/HCR1 subunit of eIF3 bridges the 40S subunit and the RRM domain in b/PRT1 to enhance eIF3 binding to 40S subunits in vivo ¹⁴.

We uncovered a novel function of the soluble ATP-binding cassette (ABC) protein RLI1 in PIC assembly, which is conserved in mammals. This essential protein is closely related to the GCN2 stimulatory factor GCN20 and translation elongation factor eEF3/YEF3. We found that RLI1 associates with eIF3, eIF5 and 40S subunits in cell extracts, and that its depletion from cells decreases translation initiation and lowers 40S occupancy of the MFC without reducing MFC integrity. Mutating the ATP binding sites in the ABCs is lethal and overexpressing one such mutant impairs translation in vivo and in cell extracts. We proposed that ATP-driven dimerization of the two ABCs could promote a conformational change in the MFC that stimulates its 40S binding ¹⁵. In collaboration with Michael Dean’s lab (NCI, NIH), we showed that the mammalian homolog of RLI1, ABCE1, interacts with eIFs 2 and 5 in vivo and that its depletion by siRNA in cells, or immunodepletion from extracts, impairs translation initiation. Consistent with this, elimination of the Xenopus homologue in embryos with antisense oligonucleotides arrests development at the gastrula stage ¹⁶.

Intrigued by the fact that RLI1, eEF3/YEF3 and GCN20 interact with ribosomes and regulate translation, we investigated the functions of the remaining soluble ABC proteins in yeast, ARB1 and NEW1. We demonstrated that ARB1 shuttles between nucleus and cytoplasm, interacts with pre-ribosomes and ribosome biogenesis factors (in both 60S and 40S branches of the pathway), and that its depletion from cells reduces the rate of pre-rRNA processing and 40S subunit levels. Deletion of NEW1 also alters 40S:60S subunit ratios ¹⁷. With our findings on RLI1, ARB1 and NEW1, it is now clear that all members of this specialized subgroup of ABC proteins are physically and functionally linked to ribosomes.

We have also initiated studies on the mechanism of 43S PIC recruitment to mRNA. Using a degron allele to deplete eIF4G1 in a strain lacking eIF4GII, we found that eliminating eIF4G greatly impairs translation initiation, as expected, but does not reduce steady-state recruitment to 40S subunits of two native mRNAs in vivo, RPL41A and MFA2. By contrast, mRNA recruitment was impaired by depleting eIF2 or eIF3 subunits. These results are surprising because it is generally assumed that eIF4G is critical for mRNA recruitment by providing a protein bridge between eIF3 (on the 40S subunit) and the factors bound to the mRNA cap (eIF4E) and poly(A) tail (PABP). Our results imply that this bridge is not essential (although could be rate-enhancing) for recruitment of at least some native mRNAs, and also that eIF2 and eIF3 can stimulate mRNA recruitment by an eIF4G-independent pathway. Depletion of eIF5 led to accumulation of these and other mRNAs in 40S PICs, providing the first in vivo evidence that its GAP function for eIF2-GTP is rate-limiting for subunit joining in vivo ⁸.

Previously, we uncovered the GCD10/TRM6 and GCD14/TRM61 genes as factors required for repression of GCN4 translation in nonstarvation conditions, and discovered that they comprise the m¹A58 tRNA methyltransferase. Lack of this modification in gcd10 or gcd14 mutants reduces tRNA_i^Met biogenesis and TC formation with attendant derepression of GCN4 ¹⁸. Work begun in my laboratory by James Anderson and Anna Krueger on suppressors of gcd10 mutations culminated (in Anderson’s lab) in discovery of a nuclear surveillance mechanism that recognizes the hypomodified tRNA_i^Met, polyadenylates it by TRF4, and targets it for degradation by the nuclear exosome ¹⁹. This was the first report describing the nuclear surveillance complex, now called TRAMP, that targets defective transcripts produced by all three RNA Polymerases.