RACK1 binding to the ribosome is required to regulate the translational efficiency of specific mRNAs

Abstract: Translation is a fundamental cellular process performed by ribosomes and is regulated by several different signaling pathways. In particular, RACK1 is a protein originally characterized as a receptor for activated PKCs and it is present on the 40S ribosomal subunit near the mRNA exit channel. RACK1 is proposed to act as an interface between signaling and ribosome machinery. To address RACK1 role in translation, we performed in vitro translation assays using HeLa lysates downregulated for RACK1. We show that RACK1 depletion impairs the translation of cap-, TOP- and stem loop-regulated mRNAs (but not IRES-dependent translation). The addition of wt exogenous RACK1 is able to rescue this translation deficit. To define if RACK1 role in translation depends by its binding to the ribosome, we generated a R36D K38E mutant RACK1 with low affinity for the ribosome. On in vitro translation, the addition of exogenous mutant RACK1 is unable to restore translation to the levels of wt RACK1. We have purified wt and mutant RACK1 from transfected HEK293 cells and interactors of both proteins are identified. Both wt and mutant RACK1 are shown to interact with wide pools of proteins, sharing several ones such as grp78. Among the interactors specific for wt RACK1, we are focusing on 40S rpSA which is a required component for 40S biogenesis. We are going to test whether rpSA presence on the ribosome is affected by RACK1 depletion, and possibly if rpSA role can contribute to explain the translation deficits in those conditions.