The eIF3a subunit drives RNA binding within the eIF3 complex

Abstract

Eukaryotic initiation factor 3 (eIF3) is a multi-subunit protein complex that binds the 40S pre-initiation complex (PIC) and has an essential role in messenger RNA (mRNA) recruitment, a process encompassing mRNA attachment and loading, scanning, and start-codon recognition. During recruitment, eIF3 is positioned to contact both the 18S ribosomal RNA (rRNA) component of the PIC and the mRNA, but how individual eIF3 subunits contribute to these RNA interactions remains poorly understood. A major obstacle has been the lack of a compositionally defined, biochemically active reconstituted yeast eIF3 complex that enables systematic analysis of individual subunits and defined subcomplexes. Here, we reconstitute Saccharomyces cerevisiae eIF3 from individually purified recombinant subunits and show that it is compositionally stable under our assay conditions and recapitulates core biochemical activities of native eIF3, including PIC binding, stimulation of capped mRNA recruitment, and RNA binding. Using this system, we show that eIF3a provides a core RNA-binding activity within eIF3 and that interactions with other eIF3 subunits modulate this activity in the intact complex and in defined eIF3a-containing subcomplexes. These results establish a reconstituted yeast eIF3 system for dissecting subunit-level biochemical activities and support a model in which subunit context tunes eIF3a-driven RNA binding during initiation.