Elucidation of the Molecular and Cellular Perturbations that Underpin the Human Disease Lethal Congenital Contracture Syndrome 1

Abstract

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 Gle1’s 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.