RACK1 on and off the ribosome

Abstract

Receptor for activated C kinase 1 (RACK1) is a eukaryote-specific ribosomal protein (RP) implicated in diverse biological functions. To engineer ribosomes for specific fluorescent labeling, we selected RACK1 as a target given its location on the small ribosomal subunit and other properties. However, prior results suggested that RACK1 has roles both on and off the ribosome, and such an exchange might be related to its various cellular functions and hinder our ability to use RACK1 as a stable fluorescent tag for the ribosome. In addition, the kinetics of spontaneous exchange of RACK1 or any RP from a mature ribosome in vitro remain unclear. To address these issues, we engineered fluorescently labeled human ribosomes via RACK1, and applied bulk and single-molecule biochemical analyses to track RACK1 on and off the human ribosome. Our results demonstrate that, despite its cellular nonessentiality from yeast to humans, RACK1 readily reassociates with the ribosome, displays limited conformational dynamics, and remains stably bound to the ribosome for hours in vitro. This work sheds insight into the biochemical basis of RPs exchange on and off a mature ribosome and provides tools for single-molecule analysis of human translation.

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

“…Third, RACK1 has extraribosomal functions in several cell lines that might be linked to transformation or only operate in specific cell types or contexts (Gallo and Manfrini, 2015;Schmitt et al, 2017). By contrast, several cell typesincluding normal cells, such as NHDFsdegrade extra-ribosomal RACK1 and restrict its function to the ribosome (Ceci et al, 2003;Gallo et al, 2018;Gerbasi et al, 2004;Jha et al, 2017;Johnson et al, 2019;Romano et al, 2019;Sengupta et al, 2004). For this reason, exogenous expression of RACK1 downregulates endogenous RACK1 levels, such that RACK1 cannot be overexpressed in these cells ( Fig.…”

Ribosomal protein RACK1 facilitates efficient translation of poliovirus and other viral IRESs

“…To investigate the function of mammalian RACK1 in translation, we established a functional rescue by expressing RACK1-FLAG in HAP1-derived CRISPR genome edited RACK1 knockout cell line RACK1 KO #1 described by Jha et al (45) using lentiviral transduction (48). HAP1 cells are a near-haploid human cell line derived from chronic myelogenous leukemia KBM-7 cells (49).…”

“…Since the 40S ribosomal subunit is not directly recruited by the PV IRES but involves a scanning mechanism, in vitro ribosome affinity can only be measured in the presence of purified translation initiation factors, which is quite challenging. However, 40S ribosomal subunits lacking RACK1 directly bind the HCV IRES with an affinity similar to wildtype 40S ribosomal subunits (48). Further, ribosomes lacking RACK1 are also able to form 80S ribosomes at high concentrations of magnesium (48) indicating that RACK1 is neither involved in 40S nor 80S:HCV IRES complex formation.…”

“…However, 40S ribosomal subunits lacking RACK1 directly bind the HCV IRES with an affinity similar to wildtype 40S ribosomal subunits (48). Further, ribosomes lacking RACK1 are also able to form 80S ribosomes at high concentrations of magnesium (48) indicating that RACK1 is neither involved in 40S nor 80S:HCV IRES complex formation. Although we cannot exclude a direct contribution of RACK1 to 40S binding or 80S complex formation with the EMCV and PV IRESs, we believe that the evidence for the HCV IRES suggests that RACK1 might employ a mechanism distinct from eS25.…”

“…While several eIF3 subunits are essential for its function, other factors such as eIF3H and eIF3J are dispensable (47, 67). Of these dispensable functions, loss of eIF3J mimics the RACK1 loss-of-function phenotype on HCV and CrPV 5′ IRES translation (47), possibly by altering the conformation of the mRNA entry channel (48). Again, the lack of a cryo-EM structure of the large PV IRES bound to the 40S ribosomal subunits prevents us from using structural data to gain insights into the mechanism of RACK1 on PV translation.…”