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Mechanistic and Transcriptome‐wide interrogation of eukaryotic translation initiation factor 3 (eIF3)
Author(s) -
Aitken Colin Echeverría,
DiNardo Acadia,
Yoo Yoon,
Stanciu Andrei
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.629.1
Subject(s) - eukaryotic initiation factor , initiation factor , biology , eukaryotic translation , translation (biology) , eif4a1 , eukaryotic small ribosomal subunit , microbiology and biotechnology , messenger rna , genetics , translational regulation , gene
Translation initiation is the rate‐limiting and most‐regulated phase of translation, and its misregulation 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. We recently probed the effects of a library of S. cerevisiae eIF3 functional variants using an in‐vitro reconstituted system. These experiments demonstrated that mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment of a native yeast mRNA. Using a series of model mRNAs to control the amount of nucleotide sequence in either the mRNA entry or exit channel, we further revealed a critical role for eIF3 in stabilizing mRNA at the exit channel. Together, these experiments are consistent with the entry‐ and exit‐channel arms of eIF3 playing distinct roles during PIC formation, mRNA recruitment, and scanning. We have also investigated the collaboration of eIF3 with Rps3/uS3 in stabilizing mRNA within the mRNA entry channel. We found that 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. More recently, we have observed that eIF3i and eIF3g contribute to the PIC‐dependent enhancement of eIF4A ATPase activity, which recent work suggests may function to remodel the mRNA entry channel. 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 genome‐wide translational effects of depleting or disrupting the eIF3 complex, using ribosome profiling. 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. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .