Molecular and Functional Dissection of a Divergent mRNA Export Pathway

About This Grant

Export of RNA from the nucleus is essential for all eukaryotic cells and has emerged as a major step in the control of gene expression. mRNA molecules are required to complete a complex series of processing events most of which are highly conserved across eukaryotes, reflecting their ancient origin, The mRNA export factor Mex67/NXF1 (yeast/vertebrates) transports its RNA cargo from the nucleus through the nuclear pore complex (NPC) to the cytoplasm on a distinct ATP dependent pathway. In most eukaryotes, Mex67/NXF1 exists as a single protein; however, additional tissue-specific isoforms of NXF1 exist in multicellular organisms (i.e. humans). However, significant deviation from the canonical pathway as described from animals and fungi has emerged in the trypanosomatids, a group of divergent unicellular parasitic protozoa. We recently discovered that Trypanosomes have three very distinct paralogs of Mex67 which we termed TbMex67, TbMex67b and TbMex67L (for Like), with differing roles in mRNA export and ribosome biogenesis. Our focus is on TbMex67 and TbMex67b, which function as general mRNA export factors, albeit also associate with paralog-specific subsets mRNA cargo and differing protein interactomes in the mammalian bloodstream form (BSF) versus the insect procyclic form (PCF) of the organism, reminiscent of the tissue-specific NXF1 variants observed in metazoa. Trypanosomatids lack individual gene promoter control, instead relying heavily on post- transcriptional gene regulation and potentially, making RNA export a crucial component in the control of gene expression. It is our hypothesis that TbMex67 and TbMex67b function to help differentially regulate the expression of genes in different life cycle stages, possibly on alternate mRNA export pathways. To determine this, we will continue to characterize the paralog-specific protein interactomes and RNA cargos of the two Mex67 proteins quantitatively and comprehensively in cells in the different life stages by utilizing and adapting methods we have pioneered, as well as standard biochemical techniques. Our preliminary work has identified putative analogs of the transcription-export complex THO/TREX which has long been considered as absent in these parasites. In addition, we have previously shown that trypanosomes depend on the Ran GTPase system, a major departure from the canonical textbook model of an ATP-dependent mRNA export machinery. We hypothesize that this system will provide a new perspective on how Ran can be utilized to mediate directional transport across the NPC. Our strategy will include classical biochemical techniques involving exogenously expressed components that have been successfully employed to delimit nucleocytoplasmic transport in yeast and humans, as well as state of the art proteomic and structural methods to compute topological maps of the TbMex67-Ran machinery. With significant implications for the mechanisms that control gene expression and hence differentiation, responses to altered environments and fitness as a parasite, these deviations may reveal additional, unsuspected, mRNA export pathways.