A critical step during gene expression is the directional export of nuclear messenger (m)RNA through nuclear pore complexes (NPCs) to the cytoplasm. During export, Gle1 in conjunction with inositol hexakisphosphate (IP6) spatially regulates the activity of the DEAD-box protein Dbp5 at the NPC cytoplasmic face. Dbp5 acts to remodel the protein composition of mRNA-protein complexes in a terminal export step. In the cytoplasm, Gle1, IP6 and Dbp5 are also required for efficient translation termination. Additionally, during translation initiation, Gle1 modulates the DEAD-box protein Ded1. GLE1 mutations are causally linked to the human disease Lethal-Congenital Contracture Syndrome 1 (LCCS-1). The main causative mutation (FinMajor) results in a three amino acid insertion (PFQ) within Gle1s essential coiled coil domain. To determine the molecular defects underlying gle1-FinMajor pathology, we analyzed the functional significance of the coiled-coil domain for human (h) and Saccharomyces cerevisiae (y) Gle1. Both yGle1 and hGle1 self-associate via their coiled coil domain in vitro to form higher order homo-oligomeric complexes. Strikingly, using electron microscopy, the hGle1 form disk-shaped structures that were malformed with the h-gle1-FinMajor protein. Because LCCS1 is a homozygous recessive condition, we established an RNAi knockdown and add-back system to test for functional defects. Reduction of GLE1 activity in HeLa cells resulted in nuclear accumulation of poly(A)+ RNA. Co-expressing siRNA-resistant wild-type hGLE1BR rescued the mRNA export defect. However, co-expression of hgle1BR-FinMajor did not. Live cell microscopy studies found that GFP-hgle1B-FinMajor had altered nucleocytoplasmic shuttling dynamics. A parallel series of genetic studies were conducted with y-gle1 loss-of-function mutants that mimic the h-gle1-FinMajor allele. Growth defects of yeast mRNA export mutants were exacerbated when combined with y-gle1-Fin alleles; whereas, translation initiation and termination mutants were not impacted. We conclude that proper Gle1 self-association is specifically required during mRNA export, revealing a new model for controlling rounds of Dbp5 activity at NPCs. This work also provides the first evidence for the molecular mechanism causing the human LCCS-1 disease, and impacts the global understanding of the role for altered mRNA transport and gene expression in other human diseases.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-04292014-111222 |
| Date | 27 May 2014 |
| Creators | Folkmann, Andrew William |
| Contributors | James Goldenring, Ronald Emeson, Ryoma Ohi, Irina Kaverina, Susan Wente |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-04292014-111222/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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