Trans­fer (t) RNAs are a major part of the trans­la­tional machin­ery and recent studies have proposed a number of mutations in enzymes involved in the modifi­ca­tion of tRNAs as being linked to human diseases. However, our knowl­edge about human tRNA modifi­ca­tions is fragmen­tary, and the most compre­hen­sive tRNA modifi­ca­tion database contain infor­ma­tion on only 20% of human tRNA modifi­ca­tions. Inter­est­ingly, tRNA thiola­tion; result­ing from intra­cel­lu­lar sulfur mobiliza­tion was found to regulate protein trans­la­tion in bacte­ria and yeast and deter­mine their responses to heat stress. Whether a similar mecha­nism also occurs in humans is not yet known. This proposal will address the hypoth­e­sis that sulfur-contain­ing amino acids in the microen­vi­ron­ment of human endothe­lial cells deter­mine tRNA thiola­tion that, in turn, regulates the trans­la­tional machin­ery during vessel devel­op­ment. The goals are to map the tRNA thio-epitran­scrip­tome for the first time, identify sulfur mobiliza­tion routes, deter­mine whether thio-modifi­ca­tions affect the trans­la­tional machin­ery and codon biased or tRNA fragmen­ta­tion processes and link the dynamic control of tRNA thiola­tion with endothe­lial cell prolif­er­a­tion. Should the hypoth­e­sis be proven correct it has the poten­tial to place the thio-epitran­scrip­tome at the center of epitran­scrip­tomic based thera­peu­tics against exces­sive vessel growth in human diseases.

Protein synthe­sis consumes enormous amounts of energy and must be tightly regulated. Numer­ous cellu­lar mecha­nisms are involved in the regula­tion of trans­la­tion, and specific modifi­ca­tions of RNA (the epitran­scrip­tome) are highly conserved among differ­ent species. Our under­stand­ing of the biolog­i­cal regula­tion and role of the epitran­scrip­tome is limited, but it is clear that dynamic modifi­ca­tions of RNA repre­sent a new layer of control of genetic infor­ma­tion. Tens of millions of RNA transcripts are present in a human cell and tRNA is the most exten­sively modified RNA species with, on an average, 13 modifi­ca­tions per molecule. Such changes are required to ensure the correct folding of tRNAs, stabi­lize the confor­ma­tion of the tRNA anticodon loop, facil­i­tate inter­ac­tions with aminoa­cyl-tRNA synthetases, and modulate decod­ing as well as ribosome process­ing. Studies to-date have focused on bacte­ria and yeast, which have devel­oped sophis­ti­cated pathways to regulate trans­la­tion in response to nutri­ents. At least in these organ­isms, the most abundant modifi­ca­tions involves de novo sulfur biosyn­the­sis which is required to thiolate tRNAs. This fasci­nat­ing mecha­nism has not yet been addressed in advanced organ­isms. A poten­tial source of highly reactive sulfur moieties for tRNA modifi­ca­tion in mammalian cells is the metab­o­lism of sulfur-contain­ing amino acids. Of these, the cysteine has been linked with sulfur trans­fer, and a post-trans­la­tional modifi­ca­tion referred to as persul­fi­da­tion or S‑sulfhydration, which is respon­si­ble for the preser­va­tion of vascu­lar homeosta­sis. Vascu­lar homeosta­sis and the transi­tion from quies­cent to prolif­er­a­tive pheno­types is a topic of inter­est in condi­tions that vascu­lar repair is impor­tant for organ­is­mal resilience mecha­nisms after injury (i.e. ischemic insults) as well as in multi­ple human diseases with increased vascu­lar angio­genic capac­ity (i.e. cancer). Although, signalling molecules and growth factors have been shown to regulate the complex morpho­genetic events involved in new blood vessel forma­tion, protein synthe­sis control mecha­nisms remain under­stud­ied. Whether, modifi­ca­tions of tRNAs and the repre­sen­ta­tion of tRNA isoac­cep­tors regulate endothe­lial cell protein synthe­sis and division is unclear. Herein, we hypoth­e­size that sulfur-contain­ing amino acids in the vascu­lar human system are the source of a novel trans­la­tion-regulat­ing code referred here as the thio-epitran­scrip­tome. Under­stand­ing whether or not the mobiliza­tion of sulfur, from sulfur-contain­ing amino acids, can impact on tRNA thio-modifi­ca­tions and subse­quently on gene expres­sion is a previ­ously unexplored topic that has the poten­tial to play a signif­i­cant role in the adapta­tion of cells to a partic­u­lar micro-environment.