In all eukaryotes, tRNAs are transcribed in the nucleus and then exported to the cytoplasm to engage in protein synthesis. However, previous work in Saccharomyces cerevisiae showed that tRNAs can also be sent back to the nucleus, and intracellular transport can be altered in response to starvation, leading to nuclear accumulation of tRNA. At least in one case, retrograde nuclear transport from the cytoplasm is necessary for wybutosine formation in tRNAPhe. Despite the fact that retrograde transport has been firmly established in yeast, it has been difficult to assess whether such a mechanism has a broader evolutionary distribution in eukaryotes.

In the first part of this dissertation, I examined the post-transcriptional modification Queuosine (Q), and its relationship to retrograde transport in Trypanosoma brucei. Q is found at the first position of the anticodon in several tRNAs (tRNATyr, tRNAAsp, tRNA Asn and tRNAHis) and is presumably important for protein synthesis, although its function is not yet fully understood. Eukaryotes cannot synthesize Q and must rely on uptake and salvage of the free base from either nutrients or from gut microbiota. Following uptake, the enzyme tRNA guanine-transglycosylase (TGT) is responsible for the incorporation of queuine into tRNA by replacing guanine. In this work I show that T. brucei's TGT (TbTGT) is a nuclear enzyme essential for Q formation in tRNA. One of its natural substrates, tRNATyr, also contains an intron, which must be removed prior to Q formation. In the present work, I show that because essential components of the splicing machinery are cytoplasmic, there is a dynamic interplay between tRNA splicing and modifications, all driven by the intracellular distribution of the different maturation components. Taken together, I demonstrate the existence of a tRNA nuclear retrograde transport pathway in T. brucei akin to what has been described in yeast, but with implications for other eukaryotic systems.

The latter half of the dissertation examines Q and its potential involvement in nutrient sensing in T. brucei. As T. brucei transitions from the procyclic insect stage, to the mammalian bloodstream stage, metabolic reprograming occurs, with concomitant changes in the expression of stage specific genes. Additionally, in T. brucei gene regulation occurs post-transcriptionally. Because of this, post-transcriptional modifications may play critical roles in regulating gene expression. Here I show that Q modification levels change between the procyclic and bloodstream developmental stages of the parasite. Q levels also fluctuate in response to changes in the availability of a subset of amino acids. These findings have implications for how organisms may use modifications to sense nutrient availability and adjust translational rates accordingly.