Molecular Mechanisms of Cue‐Induced Translation Regulation

Abstract

Precise tuning of local gene expression is essential for proper neural development. In particular, environmental cues spatially and temporally direct neuron growth by stimulating or repressing protein synthesis. The integral membrane protein, deleted in colorectal cancer, or DCC, detects the environmental growth cue netrin‐1, and transduces the growth signal to a variety of intracellular responses, including activating protein synthesis. DCC is primed to function in this way; in the absence of netrin‐1, translation machinery is tethered to its C‐terminal tail (C‐tail) and silenced. Once netrin‐1 binds DCC, the receptor dimerizes, translation machinery is released, and protein synthesis commences. How DCC's C‐tail binds to the protein synthesis machinery and prevents translation in the absence of netrin‐1 is unknown. The C‐tail is a 35 kDa protein that is composed of three domains (P1–3) and is predicted 91% unstructured. Dimerization is facilitated by the P3 domain, while the P1 domain binds to the 60S subunit protein, ribosomal protein L5 (rpL5). Titrating the full‐length C‐tail into translation‐competent lysates, we found that the tail specifically inhibits eukaryotic translation, and inhibits protein synthesis of messages that use both canonical and non‐canonical translation initiation mechanisms. Hence, we propose that the C‐tail functions primarily through binding the ribosome, not eukaryotic initiation factors. We have evidence from cosedimentation and in vitro binding studies to show that the full‐length tail binds to the small and large subunits, as well as the 80S ribosome in the absence of mRNA. Binding and luciferase reporter assays show that the P3 dimerization domain is dispensable for translation inhibition, and the P1 domain is necessary but not sufficient to bind the ribosome and decrease translation. Thus the P2 domain and/or linker sequence(s) are also important for function. Collectively, our results suggest that DCC's C‐tail inhibits translation by effectively preventing ribosomes from binding to messages. We hypothesize the unstructured C‐tail precludes mRNA binding by interacting with the decoding groove of the ribosome until receptor dimerization releases 80S ribosomes. Thus chemoattraction via netrin‐induced DCC receptor dimerization might lead to a global (not message‐specific) increase in localized protein synthesis at axonal growth cones.Support or Funding InformationMSU Denver Provost Seed GrantMSU Denver College of Letters, Arts and Sciences Dean's Seed GrantThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.