Purine-responsive expression of the Leishmania donovani NT3 purine nucleobase transporter is mediated by a conserved RNA stem-loop.

Licon, M Haley; Yates, Phillip A

The ability to modulate gene expression in response to changes in the host environment is essential for survival of the kinetoplastid parasite Leishmania. Unlike most eukaryotes, gene expression in kinetoplastids is predominately regulated post-transcriptionally. Consequently, RNA-binding proteins and mRNA-encoded sequence elements serve as primary determinants of gene regulation in these organisms; however, few have defined roles in specific stress-response pathways. Leishmania species cannot synthesize purines de novo and must scavenge these essential nutrients from the host. Leishmania have evolved a robust stress response to withstand sustained periods of purine scarcity during their lifecycles. The purine nucleobase transporter, LdNT3, is among the most substantially upregulated proteins in purine-starved Leishmania donovani parasites. Here, we report that the post-translational stability of the LdNT3 protein is unchanged in response to purine starvation. Instead, LdNT3 upregulation is primarily mediated by a 33-nucleotide-long sequence in the LdNT3 mRNA 3'-UTR that is predicted to adopt a stem-loop structure. Although this sequence is highly conserved within the mRNAs of orthologous transporters in multiple kinetoplastid species, putative stem-loops from L. donovani and Trypanosoma brucei nucleobase transporter mRNAs were not functionally interchangeable for purine-responsive regulation. Through mutational analysis of the element, we demonstrate that species specificity is attributable to just three variant bases within the predicted loop. Finally, we provide evidence that the abundance of the trans-acting factor that binds the LdNT3 stem-loop in vivo is substantially higher than required for regulation of LdNT3 alone, implying a potential role in regulating other purine-responsive genes.