Investigating the role of RNA exporter GLE1 in motor neuron disease

Emerging reports indicate that there is a strong link between mutations in genes encoding RNA-processing proteins and MNDs. The role of mRNA processing in adult and childhood forms of motor neuron disease is a major focus of research in the University. GLE1 is an mRNA export factor that is mutated in the fetal motor neuron disorder lethal congenital contracture syndrome type 1 (LCCS1) and in amyotrophic lateral sclerosis (ALS) cases. However, while significant genetic discoveries have been made in the field, patients with ALS currently have no treatment options, in part because key questions on how deficiency in RNA-binding proteins, including Gle1, causes ALS remain unanswered. Thus, research efforts aimed at understanding how mutations in these genes cause neurodegeneration are of the utmost importance to enable therapeutic development for these devastating disorders.
There is solid evidence linking Gle1 to neurodegeneration. It is however unclear how dysregulation of mRNA nuclear export, and potentially altered protein synthesis, cause the disease. We hypothesize that disruption of RNA metabolism and processing in motor neurons is a key event leading to ALS linked to mutations in the GLE1 gene.

Our research plan and objectives are therefore to:
(i) Design CRISPR- Cas9 to modulate Gle1 in motor neurons and glia cells (1-7 months)
(ii) Identify key RNA molecules with altered nuclear export in cell and iPS models (7-24 month) using cytoplasmic fractionation and human transcriptome arrays (GeneChip HTA2.0, Affymetrix): The aim is to identify all mRNA molecules that require the nuclear export activity of Gle1 in healthy cells and those affected in Gle1-mediated neurodegeneration. Data quality will initially be checked using the Expression Console software and transcripts quantified with the Transcription Analysis Console program (Affymetrix) prior to comparison with state-of-the-art tools that quantify uncertainty within PUMA. Data will further be analysed to compare accuracy as well as reproducibility according to the quality standards defined by the SEQC/MAQC-II Consortium.

(iii) Investigate the alteration of mRNP complex remodeling in disease models of Gle1. (24-36 months): under this aim, we plan to identify normal and potentially disease-altered Gle1-dependent Dbp5-remodelling of mRNPs at the proteome level. We have extensive experience with mRNP capture assays that specifically allow purification and identification of proteins directly bound to PolyA+ RNA following UV-cross-linking of live cells.