Mechanistic and Transcriptome‐wide interrogation of eukaryotic translation initiation factor 3 (eIF3)

Translation initiation is the rate‐limiting and most‐regulated phase of translation. Misregulation of translation initiation has been implicated in myriad maladies, including cancer and neurodegenerative disease. In eukaryotes, initiation is a complex pathway requiring the participation of at least twelve initiation factors (eIFs). Eukaryotic translation initiation factor 3 (eIF3) is the largest and most complex of these factors and appears to participate in events throughout the pathway.In particular, eIF3 is a pivotal player in the recruitment of mRNA to the translation pre‐initiation complex (PIC). Biochemical and genetic studies have identified interactions of eIF3 with the PIC near the mRNA entry and exit channels of the ribosome; structural studies have revealed that eIF3 binds the solvent face of the small (40S) ribosomal subunit, projecting arms into both mRNA channels and ultimately encircling the PIC.These two arms appear to play distinct roles during translation initiation. The exit channel arm has been shown to interact with mRNA sequences 5′ of the AUG and to stabilize mRNA binding at the exit channel of the PIC. While mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA, mutations in the entry channel have the most pronounced effects.eIF3 also appears to collaborate with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. Rps3/uS3 residues previously implicated in the helicase activity of the elongating ribosome stabilize mRNA binding within the ribosomal entry channel and enhance the effects of eIF3 on mRNA binding in both channels. The eIF3i and eIF3g components of the entry‐channel arm also appear to contribute to the PIC‐dependent enhancement of eIF4A ATPase activity. Recent work suggests that eIF4A may function to remodel the mRNA entry channel and thereby promote mRNA recruitment or scanning.Our current work is focused on dissecting the interactions between eIF3, the PIC, and other initiation factors in both the mRNA entry and exit channels, as well as on the inter‐subunit face of the PIC, using both in‐vitro and next‐generation sequencing approaches. In particular, we are exploring the transcriptome‐wide translational effects of depleting or disrupting the eIF3 complex, using ribosome profiling. We observe dramatic decreases in global translation for both eIF3 variants, as well as specific mRNAs whose translation is hypersensitive to each. In both cases, these mRNAs possess longer and more structured 5′ UTRs, consistent with the role of eIF3 in mRNA recruitment and scanning. In parallel, we are investigating the effects of mutations to the 40S protein Rps2 on the PIC:mRNA interaction, as well as how mutations to Rps2 and Rps3 affect the sensitivity of this interaction to mutations within the eIF3 complex.

Eukaryotic translation initiation involves a complex series of events culminating in the assembly of a ribosome containing the methionyl initiator tRNA (Met‐tRNA) positioned over the AUG start codon of an mRNA. This process is the rate‐limiting and most regulated step of translation. It begins with the formation of a pre‐initiation complex (PIC) on the small (40S) ribosomal subunit. This PIC is then recruited to the 5′ end of an mRNA, from which it scans in the 5′ to 3′ direction to identify the start (AUG) codon. This convoluted pathway is mediated by multiple protein factors called eukaryotic initiation factors (eIFs). Previous genetic and biochemical studies have shown that eIF3, the largest of these protein factors, participates in events throughout the pathway. However, characterization of eIF3's role has been challenged by this pervasive participation and its complicated architecture: eIF3 is a multisubunit complex containing at least five essential subunits, complicating mutational analysis. To circumvent this problem, we are reconstituting yeast eIF3 by recombinant expression from bacterial strains. Previous structural and biochemical studies have demonstrated the feasibility of this approach. We aim to couple it with an easily manipulatable in vitro reconstituted system to enable systematic dissection of the entire complex. Our initial studies will focus on a set of mutations targeted at understanding the role of eIF3 in mRNA recruitment: truncation of the eIF3c Nterminal domain, which has been seen to promote accurate start‐codon selection through interaction with eIF1; isolation of the eIF3a/c heterodimer that stabilizes mRNA at the ribosomal exit channel; truncation of the eIF3b C‐terminal domain, which promotes the formation of the eIF3b‐g‐i module found near the mRNA entry channel; and truncation of the eIF3b RRM domain. The effects of these mutations on component events during translation initiation will shine new light on the role of eIF3 and its subunits.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.

In the scanning model of translation initiation, the decoding site and latch of the 40S subunit must open to allow the recruitment and migration of messenger RNA (mRNA); however, the precise molecular details for how initiation factors regulate mRNA accommodation into the decoding site have not yet been elucidated. Eukaryotic initiation factor (eIF) 3j is a subunit of eIF3 that binds to the mRNA entry channel and A-site of the 40S subunit. Previous studies have shown that a reduced affinity of eIF3j for the 43S preinitiation complex (PIC) occurs on eIF4F-dependent mRNA recruitment. Because eIF3j and mRNA bind anticooperatively to the 43S PIC, reduced eIF3j affinity likely reflects a state of full accommodation of mRNA into the decoding site. Here, we have used a fluorescence-based anisotropy assay to quantitatively determine how initiation components coordinate their activities to reduce the affinity of eIF3j during the recruitment of mRNA to the 43S PIC. Unexpectedly, we show that a full reduction in eIF3j affinity for the 43S PIC requires an ATP-dependent, but unwinding-independent, activity of eIF4A. This result suggests that in addition to its helicase activity, eIF4A uses the free energy of ATP binding and hydrolysis as a regulatory switch to control the conformation of the 43S PIC during mRNA recruitment. Therefore, our results define eIF4A as a universal initiation factor in cap-dependent translation initiation that functions beyond its role in RNA unwinding.

Reconstitution and analyses of RNA interactions with eukaryotic translation initiation factors and ribosomal preinitiation complexes.

The eukaryotic 43S pre-initiation complex (PIC) containing Met-tRNAiMet in a ternary complex (TC) with eIF2-GTP scans the mRNA leader for an AUG codon in favorable "Kozak" context. AUG recognition triggers rearrangement of the PIC from an open conformation to a closed state with more tightly bound Met-tRNAiMet. Yeast ribosomal protein uS5/Rps2 is located at the mRNA entry channel of the 40S subunit in the vicinity of mRNA nucleotides downstream from the AUG codon or rRNA residues that communicate with the decoding center, but its participation in start codon recognition was unknown. We found that nonlethal substitutions of conserved Rps2 residues in the entry channel reduce bulk translation initiation and increase discrimination against poor initiation codons. A subset of these substitutions suppress initiation at near-cognate UUG start codons in a yeast mutant with elevated UUG initiation, and also increase discrimination against AUG codons in suboptimal Kozak context, thus resembling previously described substitutions in uS3/Rps3 at the 40S entry channel or initiation factors eIF1 and eIF1A. In contrast, other Rps2 substitutions selectively discriminate against either near-cognate UUG codons, or poor Kozak context of an AUG or UUG start codon. These findings suggest that different Rps2 residues are involved in distinct mechanisms involved in discriminating against different features of poor initiation sites in vivo.

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation.

The GTP-binding eukaryotic translation initiation factor eIF2 delivers initiator methionyl-tRNA to the 40 S ribosomal subunit. The factor eIF5 stimulates hydrolysis of GTP by eIF2 upon AUG codon recognition, whereas the factor eIF2B promotes guanine nucleotide exchange on eIF2 to recycle the factor for additional rounds of translation initiation. The GTP-binding (G) domain resides in the gamma subunit of the heterotrimeric eIF2; however, only eIF2beta, and not eIF2gamma, has been reported to directly bind to eIF5 or eIF2B. Using proteins expressed in yeast or recombinant systems we show that full-length yeast eIF2gamma, as well as its isolated G domain, binds directly to eIF5 and the epsilon subunit of eIF2B, and we map the interaction sites to the catalytically important regions of these factors. Consistently, an internal deletion of residues 50-100 of yeast eIF5 impairs the interaction with recombinant eIF2gamma-G domain and abolishes the ability of eIF5 to stimulate eIF2 GTPase activity in translation initiation complexes in vitro. Thus, rather than allosterically regulating eIF2gamma-G domain function via eIF2beta, our data support a model in which the GTPase-activating factor eIF5 and the guanine-nucleotide exchange factor eIF2B modulate eIF2 function through direct interactions with the eIF2gamma-G domain.

A central component in S. cerevisiae RNA polymerase I (Pol I) pre‐initiation complex (PIC) formation and transcriptional regulation is the evolutionarily conserved initiation factor termed Core Factor (CF). CF is a heterotrimer composed of Rrn6, Rrn11, and the TFIIB‐related factor Rrn7. Recent Cryo‐EM structures of the Pol I initiation complex reveal that CF resembles a right hand grasping promoter DNA in which key contacts are mediated by Rrn11 and Rrn7 (Han et al., eLife, 2017). Similar to its Pol II counterpart TFIIB, CF subunit Rrn7 consists of an N‐terminal zinc ribbon (ZR) domain followed by a linker region and two cyclin fold repeats. We systematically mutagenized the Rrn7 ZR and linker domains to further understand their role in Pol I transcription initiation. We found that ZR and linker mutants are still able to form the CF complex and assemble a PIC, yet are defective in promoter opening. These findings, paired with the Cryo‐EM structure, suggest that CF acts as a ratchet to drive promoter DNA into the active site of Pol I and that the Rrn7 ZR and linker domains play a dynamic role in ATP‐independent promoter opening.Support or Funding InformationThis work was supported by grants awarded to BAK: National Cancer Institute (5K22CA184235), Alexandrine and Alexander L. Sinsheimer Scholar award, and SUNY Research Foundation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

eIF1 Controls Multiple Steps in Start Codon Recognition during Eukaryotic Translation Initiation

One of the key roles of the 12-subunit eukaryotic translation initiation factor 3 (eIF3) is to promote the formation of the 43S and 48S pre-initiation complexes (PICs). However, particular contributions of its individual subunits to these two critical initiation reactions remained obscure. Here, we adapted formaldehyde gradient cross-linking protocol to translation studies and investigated the efficiency of the 43S and 48S PIC assembly in knockdowns of individual subunits of human eIF3 known to produce various partial subcomplexes. We revealed that eIF3d constitutes an important intermolecular bridge between eIF3 and the 40S subunit as its elimination from the eIF3 holocomplex severely compromised the 43S PIC assembly. Similarly, subunits eIF3a, c and e were found to represent an important binding force driving eIF3 binding to the 40S subunit. In addition, we demonstrated that eIF3c, and eIF3k and l subunits alter the efficiency of mRNA recruitment to 43S PICs in an opposite manner. Whereas the eIF3c knockdown reduces it, downregulation of eIF3k or eIF3l increases mRNA recruitment, suggesting that the latter subunits possess a regulatory potential. Altogether this study provides new insights into the role of human eIF3 in the initial assembly steps of the translational machinery.

Antibodies Directed against an Epitope in the N-Terminal Region of the H4L Subunit of the Vaccinia Virus RNA Polymerase Inhibit Both Transcription Initiation and Transcription Termination, in Vitro

In vitro transcription was reconstituted with HeLa cell transcription factors and RNA polymerase II, which were essentially free from DNA topoisomerase activities. DNA templates with defined negative superhelical densities were tested for transcription activity. Transcription of the Bombyx mori fibroin gene increases and plateaus from templates of increasing superhelicity, and transcription from the adenovirus 2 major late promoter rises and then falls, while transcription of the Drosophila hsp70 gene remains unchanged. Dissection of transcription into pre and post-initiation steps by the use of Sarkosyl reveals that formation of a preinitiation complex on the fibroin gene or the adenovirus 2 major late promoter is slow on relaxed DNA and accelerated by DNA superhelicity. On the contrary, the preinitiation complex assembles rapidly on the hsp70 gene irrespective of DNA topology. As is the case with the fibroin gene promoter, DNA superhelicity appears to facilitate the interaction of transcription factor IID to the adenovirus 2 major late promoter.

The human ribosome can interact with the abasic site in mRNA via a specific peptide of the uS3 protein located near the mRNA entry channel

Archaeal Translation Initiation Factor aIF2 Can Substitute for Eukaryotic eIF2 in Ribosomal Scanning during Mammalian 48S Complex Formation