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Investigation of mRNP export kinetics and gene expression regulation by the DEAD-box protein Dbp5p in Saccharomyces cerevisiae
- Author / Creator
- Lari, Azra
The intricate architecture of the eukaryotic cell, which includes the partitioning of the cell into discrete membrane bound organelles, necessitates that various cargos must cross the nuclear envelope (NE). Nuclear pore complexes (NPCs) are large macromolecular assemblies embedded within the NE that function as selective transport channels to facilitate such transport events. As part of gene expression, the export of mRNA occurs through assembly of the mRNA into a ribonucleoprotein (RNP) complex, termed an mRNP. Consequently, nuclear mRNP export requires the complex regulation and activity of dozens of RNA binding proteins (RBPs) and ~30 NPC proteins (Nups) to be successful. Yet the spatial and temporal activities of these proteins and how they determine directionality of mRNP export from the nucleus remain largely undefined. Towards understanding this complex regulation, an in vivo single particle imaging approach was employed in yeast to visualize mRNP export events with high spatial precision and temporal resolution. The resulting data reveal that mRNP export is fast (∼200 ms), and that upon arrival in the cytoplasm, mRNPs are frequently confined near the NE. Furthermore, a mutant of the principal mRNP export receptor (Mex67p) exhibits delayed cytoplasmic release from NPCs and retrograde transport of mRNPs. These results demonstrate an essential role for Mex67p in cytoplasmic mRNP release and directionality of transport, and validating the use of a live-cell yeast model to study mRNP export dynamics.
Specifically, it is the activity of an essential DEAD-box protein, Dbp5p, that is critical in driving the directional nuclear export of mRNPs. DEAD-box proteins (DBPs) are a family of RBPs found in all domains of life, often displaying RNA-stimulated ATPase activity, with diverse roles in RNA biology. Dbp5p is a dynamic shuttling protein, which accesses both the nuclear and cytoplasmic compartments. Consistent with this, activity of Dbp5p has also been linked to processes beyond mRNP export, including transcription, translation, and non-coding RNA export, suggesting the presence of distinct functional pools of Dbp5p in the nucleus and cytoplasm. However, the mechanism(s) by which nucleocytoplasmic transport occurs and how Dbp5p specifically contributes to each of these processes remain unclear. Towards understanding the functions and mechanisms of transport of Dbp5p in yeast, an alanine scanning mutagenesis was employed to generate point mutants at all possible residues within a GFP-Dbp5p reporter. Characterization of these mutants led to the identification of an N-terminal Xpo1p-dependent nuclear export signal in Dbp5p, in addition to other separation-of-function alleles. Furthermore, through characterization of mutants that alter the sub-cellular localization of Dbp5p, these data provide evidence that efficient Dbp5p nuclear shuttling is not critical for mRNP export. Rather, disruptions in Dbp5p nucleocytoplasmic transport result in tRNA export and processing defects, including changes in tRNA shuttling dynamics during recovery from nutrient stress. These data point to distinct mechanisms through which Dbp5p activity is contributing to both coding and non-coding RNP export in eukaryotic cells.
Overall, my doctoral research has provided new knowledge describing mRNP export kinetics for the first time in S. cerevisiae and a novel role for Dbp5p in tRNA processing and export. These data also provide for more accurate models of mRNA export with respect to the functions of both Mex67p and Dbp5p, and a new understanding of how Dbp5p may be centrally positioned as a global regulator of eukaryotic gene expression.
- Graduation date
- Fall 2019
- Type of Item
- Doctor of Philosophy
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