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

Abstract

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 plantbut not uncharged yeast or human loopenhances 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.

Proteomic and mechanistic dissection of the poxvirus-customized ribosome

“…We recently uncovered an unusual form of ribosome customization that occurs during infection with the poxvirus, Vaccinia Virus (VacV), which involves unique phosphorylation events in the RP RACK1 that are driven by the virus-encoded B1 kinase (Jha et al, 2017). The phosphorylated residues lie within an unstructured loop domain in RACK1 that extends into the mRNA exit channel and enhances translation of viral mRNAs, which harbor unusual poly(A)-leaders in their 5′UTRs (Jha et al, 2017;Rollins et al, 2019). Such leaders are not normally found in mammalian mRNAs, but function as cap-independent translational enhancers during poxvirus infection in part through RACK1 phosphorylation (Dhungel et al, 2017;Rollins et al, 2019;Jha et al, 2017).…”

Lipid raft associated molecules modulate signal transduction and inflammatory mediator release in mast cells

“…RACK1 is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and highly conserved throughout evolution and is found in a wide range of eukaryotic phylum from Chlorophyta to Chordata (Rollins et al, 2019;Tarnowski et al, 2014). RACK1 is expressed in all mammalian tissues and is high in brain, thymus, liver, and spleen (Wang et al, 2003).…”

“…The other loops (loops A-B and C-D) are located on the opposite side. Loop D-A between blades 6 and 7 has a flexible knob-like structure ( Figure 5A) Rollins et al, 2019;Tarnowski et al, 2014). Rollins et al, 2019).…”

“…Loop D-A between blades 6 and 7 has a flexible knob-like structure ( Figure 5A) Rollins et al, 2019;Tarnowski et al, 2014). Rollins et al, 2019). (B) RACK1 can shuttle binding partners (X) from one site to another (translocation); RACK1 could modify the activity of the partners, such as enzymes (E); RACK1 is able to change intermolecular interactions, enhancing association or dissociation of the molecules; RACK1 can modulate the stability of binding proteins, stabilizing them or promoting their degradation (Modified from Li & Xie, 2015).…”

“…RACK1 organizes these binding proteins into linear and branching signaling networks. RACK1 is thus involved in many cellular processes such as development, growth, survival, apoptosis, autophagy, translation, trafficking, morphology, adhesion and migration Cox et al, 2003;Dave et al, 2013;Jia et al, 2015;Kadrmas et al, 2007;Rollins et al, 2019;Zhao et al, 2015). It is essential that RACK1 expression be tightly regulated as any slight shift from the optimum expression level can have dramatic consequences for the organism.…”