Phosphorylation of a C-terminal RACK1 Loop by Poxviruses Controls Ribosome Translational Capacity

Rollins, Madeline Grace

doi:https://doi.org/10.21985/n2-pm64-xj06

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Phosphorylation of a C-terminal RACK1 Loop by Poxviruses Controls Ribosome Translational Capacity

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Receptor for activated C kinase 1 (RACK1) is a core small (40S) ribosomal subunit protein whose structure is highly conserved among eukaryotes apart from a C-terminal extended loop. We previously showed that a poxvirus kinase phosphorylates this flexible loop in human RACK1, mimicking endogenous negative charge in plant RACK1 to enhance translation of post-replicative poxviral transcripts with unusual polyA tracts in the 5’ untranslated region (UTR). However, the broader evolutionary significance of this loop region and the effect of a charged loop on ribosome activity remains unknown. Phylogenetic and bioinformatic analyses reveal that careful spatial organization of negative charge correlates with increased usage of 5’ polyA, but only in dicot plants and protists. RACK1 loop mutants and chimeras show that the amino acid composition of the human loop is uncharged and optimized to regulate interactions with eIF6, a eukaryotic initiation factor that controls 60S biogenesis and 80S ribosome assembly. Although in human RACK1 backgrounds 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). These data indicate that charge in the loop is the primary driver of the polyA enhancer effect, which modeling suggests operates directly on the 40S subunit. Biochemical and structural studies show that negative charge in the RACK1 loop also remodels the 40S head domain and tRNA binding sites and broadly supports non-canonical modes of translation without impacting ribotoxin-induced stress signaling and ribosome stalling on polyA tracts. Our findings unearth additional layers of translational control enabled by the RACK1 loop and uncover the immense regulatory capabilities endowed by adding a single negative charge to the loop. Collectively, our work provides a rationale for future studies exploring the extent to which specific ribosomal proteins and their corresponding post-translational modifications regulate gene expression and ribosome activity across cell types and in different species as well as the remarkable way in which species-specific functions can be mimicked in human hosts by poxviruses.

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