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Transcriptional and Translational Regulatory Mechanisms in Nutrient Control of Gene Expression

• Alan G. Hinnebusch, PhD, Head, Section on Nutrient Control of Gene Expression
• Hongfang Qiu, PhD, Staff Scientist
• Fan Zhang, MS, Senior Research Assistant
• Swati Gaikwad, PhD, Postdoctoral Fellow
• Ritu Gupta, PhD, Postdoctoral Fellow
• Rakesh Kumar, PhD, Postdoctoral Fellow
• Priyanka Mittal, PhD, Postdoctoral Fellow
• Poonam Poonia, PhD, Postdoctoral Fellow
• Priyanka Singh, PhD, Postdoctoral Fellow
• Sakshi Singh, PhD, Postdoctoral Fellow
• Vishalini Valabhoju, PhD, Postdoctoral Fellow
• Anil Vijjamarri, PhD, Postdoctoral Fellow
• Qiaoyun Zheng, PhD, Postdoctoral Fellow

We study the fundamental mechanisms involved in the assembly and function of translation initiation complexes for protein synthesis, using yeast as a model system in order to exploit its powerful combination of genetics and biochemistry. The translation initiation pathway produces an 80S ribosome bound to mRNA, with methionyl initiator tRNA (Met-tRNA_i) base-paired to the AUG start codon. The Met-tRNA_i is recruited to the small (40S) subunit in a ternary complex (TC) with the GTP–bound eukaryotic initiation factor eIF2 to produce the 43S preinitiation complex (PIC) in a reaction stimulated by eIFs 1, 1A, 3, and 5. The 43S PIC attaches to the 5′ end of mRNA, facilitated by the cap-binding complex eIF4F (comprising eIF4E, eIF4G, and the RNA helicase eIF4A) and poly(A)–binding protein (PABP) bound to the poly(A) tail, and scans the 5′ untranslated region (UTR) for the AUG start codon. Scanning is promoted by eIF1 and eIF1A, which induce an open conformation of the 40S and rapid TC binding in a conformation suitable for the scanning of successive triplets entering the ribosomal P site (P-out), and by eIF4F and other RNA helicases, such as Ded1 and its paralog Dbp1, that remove secondary structure in the 5′UTR. AUG recognition evokes tighter binding of the TC in the P-in state and irreversible GTP hydrolysis by eIF2, dependent on the GTPase–activating protein (GAP) eIF5, releasing eIF2-GDP from the PIC, with Met-tRNA_i remaining in the P site. Joining of the 60S subunit produces the 80S initiation complex ready for protein synthesis.

Our current aims in this research area are to: (1) elucidate the functions of eIF1, eIF5, eIF3, and 40S ribosomal proteins in TC recruitment and start-codon recognition; (2) identify distinct functions of the RNA helicases eIF4A (and its cofactors eIF4G/eIF4B), Ded1, and Dbp1, and of the poly(A)–binding protein (PABP) in mRNA activation, 48S PIC assembly, and scanning in vivo; (3) uncover the mechanisms of translational repression and regulation of mRNA abundance by the repressors Scd6, Pat1, the helicase Dhh1, and the mRNA–decapping enzyme Dcp2; (4) elucidate the regulation of Ded1, eIF4G, and Dhh1 functions in response to nutrient limitation or stress; and (5) elucidate the roles of the yeast orthologs of eIF2A and eIF2D in eIF2–independent initiation of translation in stress conditions.

We also analyze the regulation of amino acid–biosynthetic genes in budding yeast as a means of dissecting fundamental mechanisms of transcriptional control of gene expression. During amino acid limitation, transcription of such genes is coordinately induced by the activator Gcn4 as the result of its induction at the translational level. The eviction of nucleosomes that occlude promoter DNA sequences and block access by RNA polymerase is thought to be a rate-limiting step for transcriptional activation. Previous studies implicated certain histone chaperones, ATP–dependent chromatin-remodeling complexes, or histone acetyltransferase (HAT) complexes in eviction of promoter nucleosomes at certain yeast genes, but it is unclear whether these co-factors function at Gcn4 target genes. Our aim is to elucidate the full set of co-factors that participate in promoter nucleosome eviction at Gcn4 target genes, their involvement in this process genome-wide, and the transcriptional consequences of defective nucleosome eviction. Functional cooperation among the chromatin-remodeling complexes SWI/SNF, RSC, and Ino80, as well as the HAT complexes SAGA, NuA4, NuA3, and Rtt109/Asf1, in these processes are under study. We recently discovered that Gcn4 can activate transcription from binding sites within the coding sequences (CDS) of its target genes, inducing internal subgenic sense and antisense (AS) transcripts in addition to the conventional full-length transcripts that initiate 5′ of the CDS; and we are probing both the mechanism and possible regulatory functions of these internal AS transcripts, as well as the roles of co-transcriptional histone methylation, nucleosome reassembly, and mRNA decay enzymes in controlling their synthesis and abundance. We are also probing mechanisms involved in the asymmetric transcriptional induction of genes belonging to pairs of divergently oriented genes where only one gene responds to Gcn4 binding at the shared upstream activation sequences (enhancer).

Large-scale movement of eIF3 domains during translation initiation modulate start-codon selection.

In our previous cryo-electron microscopy (cryo-EM) reconstructions of yeast 48S preinitiation complexes (PIC), the eIF3 subcomplex (dubbed the b/i/g/a module), comprising the eIF3b subunit C-terminal domain (CTD), eIF3i, the eIF3g N-terminal domain (NTD), and an extended helical segment of eIF3a–CTD, is located near the decoding center at the 40S subunit interface, interacting with eIF1, eIF2γ, eIF3c, and the 40S itself and appearing to lock the mRNA into the 40S binding cleft. The b/i/g/a module is found at this location in both the open and closed conformations of the PIC, which are thought to depict scanning and initiation conformations, respectively, with certain contacts restricted to either the open or closed state. Surprisingly, the b/i/g/a module was found at a dramatically different position on the solvent-exposed 40S surface in our more recent py48S-5N complex, where the eIF5–NTD replaces eIF1 on the 40S subunit in a later stage of the initiation pathway. We hypothesized that, following 43S PIC attachment to mRNA, the eIF3 b/i/g/a module relocates from the solvent side to the subunit interface of the open py48S complex to help prevent PIC drop-off from mRNA during scanning, that certain of its contacts at the interface surface are remodeled on AUG recognition, and that on dissociation of eIF1 and attendant loss of eIF3b–RRM (RNA recognition motif) interaction with eIF1, the b/i/g/a module relocates to the solvent side of the 40S to allow binding of the eIF5–NTD in place of eIF1 on the 40S platform. Examining eIF3b–CTD substitutions designed to disrupt interactions of its β-propeller or RRM with eIF2γ, eIF1, or eIF3c, found uniquely at the interface surface, revealed that those conferring the strongest phenotypes increased discrimination against near-cognate UUG start codons (the Ssu^– phenotype). Binding assays confirmed the interaction of the eIF3b–RRM with eIF3c, found exclusively at the 40S subunit interface, in a manner perturbed by one such Ssu^– substitution at a predicted contact with eIF3c. Interestingly, strong Ssu^– phenotypes were also observed for eIF3b substitutions that perturb eIF3b interaction made exclusively at the solvent-exposed surface of the 40S subunit. The findings suggest that interactions of the b/i/g/a module with certain initiation factors at the subunit interface act primarily to stabilize the closed conformation of the PIC on start-codon recognition, that relocation of the module back to the solvent interface is required to finalize start-codon selection, and that these interactions are crucial for the ability to utilize non-cognate initiation codons in vivo.

Amino acid residues of 40S protein uS5/Rps2 at the mRNA entry channel enhance initiation at suboptimal start codons in vivo.

The ribosomal protein uS3/Rps3 is positioned at the solvent side of the 40S near the mRNA entry channel. We showed previously that substituting uS3/Rps3 residues that contact mRNA preferentially destabilizes the closed conformation of the PIC, reducing initiation at both UUG codons and at AUG start codons that reside in suboptimal ‘Kozak’ sequence (5′-(gcc)gccRccAUGG-3′) context (a sequence regarded as the optimum sequence for initiating translation in eukaryotes). Particular residues of uS5/Rps2 make distinct mRNA contacts at the 40S entry channel and also interact with rRNA elements that communicate with the 40S decoding center. We found that uS5/Rps2 substitutions V121D and I125K resemble the previously characterized uS3/Rps3 substitutions in suppressing initiation at UUG codons as well the poor-context AUG start codons in SUI1 mRNA or an elongated form of upstream open-reading frame 1 of GCN4 mRNA (el.-uORF1). Interestingly, the uS5/Rps2 substitutions D78A, Q89K, and K119A suppress UUG initiation but do not reduce initiation at the poor-context AUG codons, and thus appear to be specific for suppressing near-cognate initiation. Substitutions Q94D and T96K diminish initiation at the poor-context AUG codons of SUI1 and el.-uORF1, and they efficiently suppress UUG initiation only when the UUG resides in poor sequence context. Thus, the latter two residues appear to act mainly in discriminating against poor Kozak context. The findings suggest that different uS5/Rps2 residues are involved in distinct mechanisms of discrimination against different features of poor initiation sites in vivo.

Reprogramming of translation in yeast cells impaired for ribosome recycling favors short, efficiently translated mRNAs.

In eukaryotes, formation of the 43S PIC, containing initiator Met-tRNA_i bound to the small ribosomal subunit, is a rate-determining step of translation initiation. Ribosome recycling after translation termination produces the free 40S subunits needed to reassemble 43S PICs for new initiation events. Yeast mutants lacking orthologs of mammalian eIF2D (Tma64), and either MCT-1 (Tma20) or DENR (Tma22), are broadly impaired for 40S recycling; however, it was unknown whether the defect alters the translational efficiencies (TEs) of mRNAs. Based on previous experiments, it was also possible that Tma64/eIF2D can substitute for eIF2 in recruitment of Met-tRNA_i during initiation. Consistent with impaired initiation, the tma64Δtma20Δ mutant exhibits reduced assembly of bulk polysomes. Ribosome profiling of this mutant reveals a marked reprogramming of translational efficiencies, wherein translation of the most efficiently translated (‘strong’) mRNAs tends to be elevated, whereas translation of ‘weak’ mRNAs generally declines. Profiling of the tma64Δ single mutant reveals none of the hallmarks of impaired 40S recycling nor changes in translation efficiencies, suggesting that the defects found in tmaΔΔ cells are associated with defective ribosome recycling rather than with loss of eIF2D function in Met-tRNA_i recruitment. Remarkably, we observed similar translational re-programming on reducing 43S PIC assembly by inducing phosphorylation of eIF2 or by decreasing total 40S subunit levels by depleting Rps26, without affecting ribosome recycling. Moreover, the tmaΔΔ mutation specifically impaired translation of mRNAs with cap-proximal secondary structures that are expected to impede PIC attachment. Our findings suggest that strong mRNAs outcompete weak mRNAs in response to 43S PIC limitation achieved in various ways at the step of 43S PIC recruitment, in accordance with mathematical modeling of how translational efficiencies of different groups of mRNAs are altered by reduced ribosome abundance. The findings also have important implications for understanding changes in translation occurring in human ribosomopathies in which 40S subunit levels are diminished.

Down-regulation of yeast helicase Ded1 by glucose starvation or heat-shock differentially impairs translation of Ded1–dependent mRNAs.

Ded1 is an essential DEAD-box helicase in yeast that broadly stimulates translation initiation and is critical for mRNAs with structured 5′UTRs. Recent evidence suggests that the condensation of Ded1 in mRNA granules down-regulates Ded1 function during heat-shock or glucose starvation. We examined this hypothesis by determining the overlap between mRNAs whose relative translational efficiencies (TEs), as determined by ribosomal profiling, were diminished in either stressed wild-type (WT) cells or in ded1 mutants examined in non-stress conditions. Only subsets of the Ded1–hyperdependent mRNAs identified in ded1–mutant cells exhibited strong TE reductions in glucose-starved or heat-shocked WT cells; and those down-regulated by glucose starvation also exhibited hyper-dependence on the initiation factor eIF4B, and to a lesser extent of eIF4A, for efficient translation in non-stressed cells. The findings are consistent with recent proposals that the dissociation of Ded1 from mRNA 5′UTRs and the condensation of Ded1 contribute to reduced Ded1 function during stress, and they further suggest that the down-regulation of eIF4B and eIF4A functions also contributes to the translational impairment of a select group of Ded1 mRNA targets with heightened dependence on all three factors during glucose starvation.

Distinct functions of three chromatin remodelers in activator binding and preinitiation-complex (PIC) assembly

The nucleosome-remodeling complexes (CRs) SWI/SNF, RSC, and Ino80C cooperate in evicting or repositioning nucleosomes to produce nucleosome-depleted regions (NDRs) at the promoters of many yeast genes induced by amino acid starvation. We analyzed mutants lacking the CR catalytic subunits for binding of the transcriptional activator Gcn4 and recruitment of TATA–binding protein (TBP) during PIC assembly. RSC and Ino80 enhance Gcn4 binding to UAS (upstream activation sequence) elements in NDRs upstream of many promoters as well as to unconventional binding sites within nucleosome-occupied coding sequences; and SWI/SNF contributes to UAS binding when RSC is depleted. All three CRs are actively recruited by Gcn4 to most UAS elements and appear to enhance Gcn4 binding by reducing nucleosome occupancies at the binding motifs, indicating a positive regulatory loop (Figure 1). SWI/SNF acts unexpectedly in WT cells to prevent excessive Gcn4 binding at certain UAS elements, which might involve transient nucleosome sliding that does not alter steady-state nucleosome occupancies. All three CRs also stimulate TBP recruitment, at least partly by reducing nucleosome occupancies at TBP binding sites, with SWI/SNF acting preferentially at the most highly expressed Gcn4 target genes. RSC and Ino80 function more broadly than SWI/SNF to stimulate TBP recruitment at most constitutively expressed genes, including ribosomal protein genes, whereas SWI/SNF acts preferentially at a distinct subset of highly expressed genes. Our findings point to a complex interplay among the three CRs in evicting promoter nucleosomes to regulate activator binding and stimulate PIC assembly.

Publications

1. Dong J, Hinnebusch AG. uS5/Rps2 residues at the 40S ribosome entry channel enhance initiation at suboptimal start codons in vivo. Genetics 2022 220:iyab176.
2. Llácer JL, Hussain T, Dong J, Villamayor L, Gordiyenko Y, Hinnebusch AG. Large-scale movement of eIF3 domains during translation initiation modulate start codon selection. Nucleic Acids Res 2021 49(20):11491–11511.
3. Gaikwad S, Ghobakhlou F, Young DJ, Visweswaraiah J, Zhang H, Hinnebusch AG. Reprogramming of translation in yeast cells impaired for ribosome recycling favors short, efficiently translated mRNAs. eLife 2021 10:e64283.
4. Sen ND, Zhang H, Hinnebusch AG. Down-regulation of yeast helicase Ded1 by glucose starvation or heat-shock differentially impairs translation of Ded1-dependent mRNAs. Microorganisms 2021 9:2413.

Collaborators

• Jose L. Llácer, PhD, Instituto de Biomedicina de Valencia (IBV-CSIC), Valencia, Spain
• Jon Lorsch, PhD, Laboratory on the Mechanism and Regulation of Protein Synthesis, NICHD, Bethesda, MD
• Venkatraman Ramakrishnan, PhD, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
• Neelam Sen, PhD, Jawaharlal Nehru University, New Delhi, India
• Hussain Tanweer, PhD, Indian Institute of Science, Bangalore, India

Contact

For more information, email hinnebua@mail.nih.gov or visit https://www.nichd.nih.gov/research/atNICHD/Investigators/hinnebusch.

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