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Molecular mechanisms and inhibition of mRNA recruitment in eukaryotic and viral translation initiation

  • Author(s): Berry, Katherine Elizabeth
  • Advisor(s): Doudna, Jennifer A
  • et al.
Abstract

The regulation of protein synthesis is critical in viral infection, cell death, and development. Most regulation of translation occurs during the rate-limiting step of translation initiation, the process by which the ribosome binds to and positions initiator tRNA and messenger RNA at the initiation (AUG) codon. An understanding of the detailed mechanisms of this initiation process is critical to our understanding of translational control in biology. This work discusses the molecular mechanisms by which messenger RNA is recruited to human 40S ribosomal subunits during translation initiation and positioned correctly in the mRNA binding cleft and our efforts to inhibit this process in the lifecycle of Hepatitis C virus.

Eukaryotic initiation factor 3 (eIF3) was previously thought to bind primarily to the solvent side of 40S ribosomal subunits, and the principal role of the eIF3j subunit during translation initiation was believed to be facilitating eIF3 binding to 40S subunits. The C-terminal domain of human eIF3j is now known to bind in the mRNA binding cleft and aminoacyl (A) site of the 40S ribosomal subunit. We utilized a recombinant biochemical system of human initiation factors to biophysically examine eIF3j's interaction with mRNA and interface binding factors to define the role of this eIF3 subunit in the translation initiation pathway. We demonstrate that eIF3j interacts directly or indirectly with eIF1A on the 40S subunit. We also show that eIF3j influences the interaction of mRNA with the 40S subunit's mRNA binding cleft during translation initiation, reducing mRNA's affinity for 40S subunits until eIF2-tRNAimet-GTP (TC) is present. These biochemical observations help to explain why deletion of eIF3j from S. cerevisiae leads to a leaky scanning phenotype.

Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. The HCV genomic RNA contains an internal ribosome entry site (IRES) in its upstream untranslated region (UTR), the structure of which is essential for viral protein translation. We developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV IRES. Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a capped mRNA control. Despite well-optimized in vitro translation conditions, no selective HCV IRES inhibitors were found in the end, as the vast majority of hits proved to be luciferase and general translation inhibitors. The analysis of these molecules, and the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors.

The HCV IRES faces a challenge in that the majority of its structure binds to the solvent side of the 40S subunit, yet its initiation codon needs to reach the P site in the mRNA binding cleft, and must do so without the many cap-binding and scanning factors used by a cellular message. The IRES includes a predicted pseudoknot interaction near the AUG start codon, but the results of previous studies of its structure have been conflicting. Using mutational analysis coupled with activity and functional assays, we verified the importance of pseudoknot base pairings for IRES-mediated translation, and conducted a comprehensive study of the structural tolerance and functional contributions of the pseudoknot. Ribosomal toeprinting experiments show that the entirety of the pseudoknot element positions the initiation codon in the mRNA binding cleft of the 40S ribosomal subunit. Optimal spacing between the pseudoknot and the start site AUG resembles that between the Shine-Dalgarno sequence and the initiation codon in bacterial mRNAs. In addition, we validated the HCV IRES pseudoknot as a potential drug target using antisense oligonucleotides. Initial steps have been taken to solve a high-resolution structure of this IRES pseudoknot domain.

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