We employ a suite of biochemical, biophysical, and genome-scale approaches to investigate how the multi-subunit eIF3 complex contributes to the mechanism of translation initiation and to translational regulation.

An in vitro reconstituted translation initiation system

We employ a S. cerevisiae reconstituted in vitro translation initiation system. This system is composed of purified initiation factors (eIFs 1, 1A, 2, 3, 4A, 4B, 4G/E, 5, and PABP), ribosomal subunits, in vitro transcribed and methionylated tRNAi, and in vitro transcribed and capped mRNA. These components enable a broad repertoire of binding and activity assays that report on component events and interactions of the translation initiation pathway, including an assay developed by Dr. Aitken (during his postdoctoral fellowship in the laboratory of Dr. Jon Lorsch) that probes interactions between the ribosomal pre-initiation complex (PIC) and mRNA in the mRNA-entry- and mRNA-exit channels of the ribosome. We also make use of a library of purified and previously characterized eIF3 functional variants This library—which Dr. Aitken also established as a postdoctoral fellow to investigate the mRNA-entry-channel and mRNA-exit-channel arms—contains mutations distributed throughout the eIF3 complex and affecting the diverse functional roles of eIF3. Together, these tools represent a unique and powerful system for mechanistic interrogation of eIF3.

An in vitro reconstituted eIF3 complex

Genetic studies have identified lethal mutations throughout the eIF3 complex However, because each of the core eIF3 subunits is essential and previous in vitro analyses have relied on eIF3 purified from the endogenous yeast pool, these studies have been limited to the investigation of viable mutants. The inability to interrogate lethal variants, sub-complexes, and subunits or specifically target regions of eIF3 shown to interact with other factors in vitro represents a fundamental obstacle in the field. Building on previously reported but functionally uncharacterized approaches, we have developed the eIF3^rec system, which recapitulates the functions of the natively purified complex (eIF3^nat), enabling the full dissection of the eIF3 complex with an array of ensemble and single-molecule biophysical techniques.  

Recently posted:  Ide & Gentry, et al., bioRxiv 2024

Interrogating the genome-scale roles of eIF3

Beyond its critical roles in the mechanism of translation initiation, eIF3 has more recently been implicated in translational regulation. However, the mechanism(s) whereby eIF3 can mediate distinct translational fates for specific mRNAs remain mysterious. We employ a portfolio of genome-scale tools, including ribosome profiling, tandem-mass-tag mass spectrometry (TMT-MS) as well as both traditional RNA sequencing and long-read nanopore sequencing of mRNA pools fractionated according to their translational status. Using ribosome profiling, we have demonstrated that severe disruption of either the entire eIF3 complex or its mRNA-entry-channel arm affect the translation of mRNAs across the transcriptome. Notably, mRNAs with long and structured 5′-untranslated regions (5′-UTRs) are most sensitive to these disruptions, as are mRNAs most sensitive to mutations targeting other eIFs involved in the mRNA activation and scanning steps of initiation. Our experiments also reveal that eIF3 may play regulatory roles even in S. cerevisiae, suggesting that its regulatory roles in higher eukaryotes may have originated in simpler eukaryotes. Using long-read nanopore sequencing, we are investigating the specific 5′-UTRs sequences underlying these effects.

Recently published:  Stanciu, et al., Front. Mol. Biosci. 2022

Recently posted:  Koubek, et al., bioRxiv 2021