Abstract
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 posttranscriptionally. 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 life cycle. The purine nucleobase transporter LdNT3 is among the most substantially up-regulated proteins in purine-starved Leishmania donovani parasites. Here we report that the posttranslational stability of the LdNT3 protein is unchanged in response to purine starvation. Instead, LdNT3 up-regulation 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.
Highlights
The ability to modulate gene expression in response to changes in the host environment is essential for survival of the kinetoplastid parasite Leishmania
It has been published that changes in both mRNA stability and translational efficiency contribute to LdNT3 up-regulation under purine starvation [16, 18]
When probing the purine stress response, we used UMP synthase (UMPS) as an unresponsive control because neither UMPS mRNA nor protein abundance are affected by purine starvation [16, 18]
Summary
We show that the LdNT3 stem–loop is sufficient to confer purine responsiveness to a high-abundance transcript, suggesting that the cognate RBP responsible for binding this element in vivo is present within the cell in substantial excess of what is required to regulate LdNT3 expression alone. To test whether this region confers purine responsiveness in L. donovani, we generated cell lines in which a Fluc-BSD transgene was expressed from the endogenous LdNT3 locus under the control of WT UTRs or a modified 3Ј UTR lacking the putative stem–loop (Fig. 2B) Parasites expressing Fluc-BSD flanked by WT UTRs demonstrated an ϳ27-fold increase in luciferase activity in response to purine stress.
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