One protein many functions: the non-canonical interactions of SHMT1. The structural and functional characterization of SHMT1 interactions with RNA and in the de novo thymidylate synthesis complex

Several enzymes that were originally characterized to have one defined function in intermediatory metabolism, have now been found to participate in variety of other cellular processes 1,2. In fact, some metabolic enzymes, named moonlighting proteins, have been found to non-canonically interact with other proteins or nucleic acids. Interestingly, not all the newly found RNA binding proteins have been shown to regulate the nucleic acid’s function, identifying a novel process known as riboregulation. The catalytic activity of human serine hydroxymethyltransferase (SHMT1) has been found to be controlled by RNA 3. Moreover, it has been demonstrated that this enzyme takes part in the assembly of the thymidylate synthesis complex (dTMP-SC) in the nucleus, together with thymidylate synthase (TYMS) and dihydrofolate reductase (DHFR) 4. Therefore, the aim of the present thesis is to investigate the details of the non-canonical functions of SHMT1, including the molecular mechanism at the basis of SHMT1 riboregulation. Here I will present the first 3D structure of a metabolic enzyme in complex with RNA, solved using cryo-election microscopy (cryo-EM), and the identification of the RNA features governing the interaction with SHMT1. In parallel, I will present a deep biochemical and molecular characterization of the dTMP-SC, providing the evidence that the complex can transiently form in vitro and in the cytosol as well. An observation that opens a novel scenario on its relevance in different processes. The present manuscript set the basis to fully understand how metabolic enzymes can be controlled by RNA and strengthen the idea that transient interactions might regulate the organization of biomolecular function in response to the cellular needs. 1 P. Huangyang, M.C. Simon, Hidden features: Exploring the non- canonical functions of metabolic enzymes, DMM Dis. Model. Mech. 11 (2018). https://doi.org/10.1242/dmm.033365. 2 A. Castello, M.W. Hentze, T. Preiss, Metabolic Enzymes Enjoying New Partnerships as RNA-Binding Proteins, Trends Endocrinol. Metab. 26 (2015) 746–757. https://doi.org/10.1016/j.tem.2015.09.012. 3 G. Guiducci, A. Paone, A. Tramonti, G. Giardina, S. Rinaldo, A. Bouzidi, M.C. Magnifico, M. Marani, J.A. Menendez, A. Fatica, A. MacOne, A. Armaos, G.G. Tartaglia, R. Contestabile, A. Paiardini, F. Cutruzzol, The moonlighting RNA-binding activity of cytosolic serine hydroxymethyltransferase contributes to control compartmentalization of serine metabolism, Nucleic Acids Res. 47 (2019) 4240–4254. https://doi.org/10.1093/nar/gkz129. 4 D. Anderson, C. Quintero, P. Stover, Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria, Proc. Natl. Acad. Sci. U. S. A. 108 (2011) 15163–15168. https://doi.org/10.1073/PNAS.1103623108.