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Investigating the Role of Rps2 in Pre‐Initiation Complex Stability Using an In Vitro Assay for mRNA Recruitment
Author(s) -
DiNardo Acadia L,
Aitken Colin Echeverría
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.629.2
Subject(s) - biology , eukaryotic initiation factor , initiation factor , messenger rna , microbiology and biotechnology , translation (biology) , eukaryotic translation , translational regulation , genetics , gene
The translation of mRNA into protein is highly regulated, with misregulation often observed in viral infections, cancer, neurodegenerative disease, and other maladies. Translation initiation is the most regulated step of this process, and requires the participation of at least twelve protein initiation factors in eukaryotic cells. Eukaryotic translation initiation factor 3 (eIF3) plays a central role in recruitment of mRNA to the pre‐initiation complex (PIC) and is known to interact with the PIC near the mRNA entry and exit channels of the ribosome. Structural analysis has shown that eIF3 binds to the solvent face of the small ribosomal subunit (40S) and projects arms into each of these channels, surrounding the PIC. Previous research has analyzed the effects of mutations to the eIF3 complex on discrete interactions and events during initiation utilizing an in vitro reconstituted system. These studies revealed that mutations to eIF3 disrupt PIC interactions and affect the rate and efficiency of mRNA recruitment. mRNA recruitment experiments with model mRNAs that control the number of nucleotides present in both the entry and exit channels of the PIC demonstrate that eIF3 plays a critical role in the stabilization of mRNA in the exit channel. These experiments also suggest that eIF3 may contribute to the mechanistic events underlying the rate of recruitment at the entry channel. Consistent with previous work, this contribution may involve collaboration with the 40S latch elements Rps2/3. Indeed, mutations to Rps3 destabilize mRNA bound by the PIC in a manner that exacerbates its dependence on eIF3 for this interaction. We are now interrogating the effect of mutations to Rps2 within a similar in vitro reconstituted system using five distinct model mRNAs. Previous work has identified Rps2 mutants that may affect conformational changes of the PIC in response to mRNA binding and start‐codon recognition, and thereby destabilize its binding to mRNA. We are now investigating the effects of these mutations on mRNA recruitment. We will also further interrogate the interaction between eIF3 and the 40S latch by asking whether mutations to Rps2 or Rps3 are sensitive to mutations within elements of eIF3 thought to interact the latch. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .