RACK1 evolved species-specific multifunctionality in translational control through sequence plasticity within a loop domain

Receptor of activated protein C kinase 1 (RACK1) is a highly conserved eukaryotic protein that regulates several aspects of mRNA translation; yet, how it does so, remains poorly understood. Here we show that, although RACK1 consists largely of conserved β-propeller domains that mediate binding to several other proteins, a short interconnecting loop between two of these blades varies across species to control distinct RACK1 functions during translation. Mutants and chimeras revealed that the amino acid composition of the loop is optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Separately, phylogenetics revealed that, despite broad sequence divergence of the loop, there is striking conservation of negatively charged residues amongst protists and dicot plants, which is reintroduced to mammalian RACK1 by poxviruses through phosphorylation. Although both charged and uncharged loop mutants affect eIF6 interactions, only a negatively charged plant - but not uncharged yeast or human loop - enhances translation of mRNAs with adenosine-rich 5′ untranslated regions (UTRs). Our findings reveal how sequence plasticity within the RACK1 loop confers multifunctionality in translational control across species.