Investigating the role of SERBP1 in translation

Idle ribosomes are inactive 80S monosomes that lack mRNA and are common throughout eukaryotes. In yeast, they promote starvation recovery and translational reactivation. In metazoan, their physiological functions and the mechanisms governing their abundance are unclear. Current hypotheses posit that they play a role in fine tuning protein synthesis under conditions requiring tight regulation such as cell development and plasticity. Previously, we determined cryo-EM structures of idle ribosomes containing serpine 1 mRNA binding protein (SERBP1) and phosphorylated eukaryotic elongation factor 2 (p-eEF2). SERBP1 binds to the 40S head via a small helix and threads through the mRNA channel, where it interacts with eEF2. Here, we term these regions ‘anchor’ and ‘thread’, respectively. Stimulation of eEF2 kinase (eEF2K) increases the abundance of SERBP1/p-eEF2 idle ribosomes and prevents their recycling in vitro. SERBP1 also regulates the stability of specific mRNAs and contains motifs that drive phase separation. We propose that different regions of SERBP1 mediate its functions in translation and localization in liquid phase separation. We designed structure-informed truncations and mutations and examined their effect on protein synthesis in an in vitro translation system. We found that recombinant full-length SERBP1 strongly inhibits translation. A C-terminal truncation including the thread and anchor regions shows a similar inhibitory effect, attenuated upon mutating the binding sites with eEF2. The SERBP1 anchor alone does not affect protein synthesis. eEF2K strongly inhibits translation and this effect is not enhanced in the presence of the anchor. Collectively, our data suggest that the SERBP1 thread and specifically the residues interacting with eEF2 are critical for attenuation of protein synthesis, likely by promoting the formation of idle ribosomes. Further studies will illuminate if other SERBP1 regions are implicated in this process and in phase separation, and if eEF2K acts synergistically to inhibit translation.