GCD14p, a repressor of GCN4 translation, cooperates with Gcd10p and Lhp1p in the maturation of initiator methionyl-tRNA in Saccharomyces cerevisiae.

Gcd10p and Gcd14p were first identified genetically as repressors of GCN4 mRNA translation in Saccharomyces cerevisiae. Recent findings indicate that Gcd10p and Gcd14p reside in a nuclear complex required for the presence of 1-methyladenosine in tRNAs. Here we show that Gcd14p is an essential protein with predicted binding motifs for S-adenosylmethionine, consistent with a direct function in tRNA methylation. Two different gcd14 mutants exhibit defects in cell growth and accumulate high levels of initiator methionyl-tRNA (tRNAiMet) precursors containing 5' and 3' extensions, suggesting a defect in processing of the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNAiMet (hcIMT1 to hcIMT4; hc indicates that the gene is carried on a high-copy-number plasmid) or a homologue of human La protein implicated in tRNA 3'-end formation (hcLHP1), also suppressed gcd14 mutations. In fact, the lethality of a GCD14 deletion was suppressed by hcIMT4, indicating that the essential function of Gcd14p is required for biogenesis of tRNAiMet. A mutation in GCD10 or deletion of LHP1 exacerbated the defects in cell growth and expression of mature tRNAiMet in gcd14 mutants, consistent with functional interactions between Gcd14p, Gcd10p, and Lhp1p in vivo. Surprisingly, the amounts of NME1 and RPR1, the RNA components of RNases P and MRP, were substantially lower in gcd14 lhp1::LEU2 double mutants than in the corresponding single mutants, whereas 5S rRNA was present at wild-type levels. Our findings suggest that Gcd14p and Lhp1p cooperate in the maturation of a subset of RNA polymerase III transcripts.

Inhibition of double-stranded RNA-dependent protein kinase PKR by vaccinia virus E3: role of complex formation and the E3 N-terminal domain.

The human double-stranded RNA (dsRNA)-dependent protein kinase PKR inhibits protein synthesis by phosphorylating translation initiation factor 2alpha (eIF2alpha). Vaccinia virus E3L encodes a dsRNA binding protein that inhibits PKR in virus-infected cells, presumably by sequestering dsRNA activators. Expression of PKR in Saccharomyces cerevisiae inhibits protein synthesis by phosphorylation of eIF2alpha, dependent on its two dsRNA binding motifs (DRBMs). We found that expression of E3 in yeast overcomes the lethal effect of PKR in a manner requiring key residues (Lys-167 and Arg-168) needed for dsRNA binding by E3 in vitro. Unexpectedly, the N-terminal half of E3, and residue Trp-66 in particular, also is required for anti-PKR function. Because the E3 N-terminal region does not contribute to dsRNA binding in vitro, it appears that sequestering dsRNA is not the sole function of E3 needed for inhibition of PKR. This conclusion was supported by the fact that E3 activity was antagonized, not augmented, by overexpressing the catalytically defective PKR-K296R protein containing functional DRBMs. Coimmunoprecipitation experiments showed that a majority of PKR in yeast extracts was in a complex with E3, whose formation was completely dependent on the dsRNA binding activity of E3 and enhanced by the N-terminal half of E3. In yeast two-hybrid assays and in vitro protein binding experiments, segments of E3 and PKR containing their respective DRBMs interacted in a manner requiring E3 residues Lys-167 and Arg-168. We also detected interactions between PKR and the N-terminal half of E3 in the yeast two-hybrid and lambda repressor dimerization assays. In the latter case, the N-terminal half of E3 interacted with the kinase domain of PKR, dependent on E3 residue Trp-66. We propose that effective inhibition of PKR in yeast requires formation of an E3-PKR-dsRNA complex, in which the N-terminal half of E3 physically interacts with the protein kinase domain of PKR.

Dimerization by translation initiation factor 2 kinase GCN2 is mediated by interactions in the C-terminal ribosome-binding region and the protein kinase domain.

The protein kinase GCN2 stimulates translation of the transcriptional activator GCN4 in yeast cells starved for amino acids by phosphorylating translation initiation factor 2. Several regulatory domains, including a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome association, have been identified in GCN2. We used the yeast two-hybrid assay, coimmunoprecipitation analysis, and in vitro binding assays to investigate physical interactions between the different functional domains of GCN2. A segment containing about two thirds of the protein kinase (PK) catalytic domain and another containing the C-term region of GCN2 interacted with themselves in the two-hybrid assay, and both the PK and the C-term domains could be coimmunoprecipitated with wild-type GCN2 from yeast cell extracts. In addition, in vitro-translated PK and C-term segments showed specific binding in vitro to recombinant glutathione S-transferase (GST)-PK and GST-C-term fusion proteins, respectively. Wild-type GCN2 could be coimmunoprecipitated with a full-length LexA-GCN2 fusion protein from cell extracts, providing direct evidence for dimerization by full-length GCN2 molecules. Deleting the C-term or PK segments abolished or reduced, respectively, the yield of GCN2-LexA-GCN2 complexes. These results provide in vivo and in vitro evidence that GCN2 dimerizes through self-interactions involving the C-term and PK domains. The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the HisRS domain interacted with the C-term region. We propose that physical interactions between the PK domain and its flanking regulatory regions and dimerization through the PK and C-term domains both play important roles in restricting GCN2 kinase activity to amino acid-starved cells.

Autophosphorylation in the activation loop is required for full kinase activity in vivo of human and yeast eukaryotic initiation factor 2alpha kinases PKR and GCN2.

The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit alpha (eIF2alpha) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2alpha kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop.

GCD10, a translational repressor of GCN4, is the RNA-binding subunit of eukaryotic translation initiation factor-3.

GCN4 mRNA is translated by a reinitiation mechanism involving four short upstream open reading frames (uORFs) in its leader sequence. Decreasing the activity of eukaryotic initiation factor-2 (eIF-2) by phosphorylation inhibits general translation in yeast but stimulates GCN4 expression by allowing ribosomes to scan past the uORFs and reinitiate at GCN4 instead. GCD10 was first identified genetically as a translational repressor of GCN4. We show here that GCD10 is an essential protein of 54.6 kD that is required in vivo for the initiation of total protein synthesis. GCD10 binds RNA in vitro and we present strong biochemical evidence that it is identical to the RNA-binding subunit of yeast initiation factor-3 (eIF-3). eIF-3 is a multisubunit complex that stimulates translation initiation in vitro at several different steps. We suggest that gcd10 mutations decrease the ability of eIF-3 to stimulate binding of eIF-2/GTP/Met-tRNA(iMet) ternary complexes to small ribosomal subunits in vivo. This would explain why mutations in eIF-3 mimic eIF-2 alpha phosphorylation in allowing ribosomes to bypass the uORFs and reinitiate at GCN4. Our results indicate that GCN4 expression provides a sensitive in vivo assay for the function of eIF-3 in initiation complex formation.