Amyotrophic Lateral Sclerosis (ALS) is a devastating disease that results in progressive immobility and death of affected individuals. Paralysis is driven by the degeneration of motor neurons (MNs), the nerve cells that innervate muscle. Complex disease heterogeneity and incomplete understanding of the mechanisms driving ALS has resulted in poor therapeutic options. However, recent advances have uncovered common disruption to RNA processing events including: transcription, splicing, transport, translation and post-transcriptional repression. The mechanisms behind this dysregulation have yet to be fully elucidated.
This thesis interrogates the roles of RNA regulators in ALS. We focus our attention on microRNAs (miRs) and RNA binding proteins (RBPs). MiRs are post-transcriptional regulators of gene expression, and are commonly disrupted in ALS. Using transcriptomic and bioinformatic analysis, we identified that miR-139 is downregulated in a majority of ALS cases. Exploration into the mechanism of action of this miR led us to discover a feedback loop between miR-139 and a key signalling pathway called WNT. We found that disruption of this feedback loop leads to WNT upregulation and MN degeneration in ALS.
Transcriptomic analysis of patient tissue has revealed several microRNAs downregulated in ALS. As different miRs can converge on similar genes and/or pathways, we explored the role of ALS-miRs by inhibiting 44 microRNAs and analysing the downstream effects on cellular phenotypes. By conducting pathway analysis on their downstream targets, we uncovered convergence of ALS-associated miRs on RNA metabolism pathways, indicating their contribution to global disruption of RNA processing events. This also indicated that regulation of RBPs was occurring at the post-transcriptional level, altering protein expression.
Completing protein screens is technically more difficult than genomic and transcriptomic analysis, due to the limited technology available. Current methods to assess RBP expression, activity and/or targets are low-throughput. To circumvent these limitations, we developed a novel method to obtain a holistic view of RBP-RNA interactions. Exonuclease-assisted mapping of protein-RNA interactions (ePRINT) captures a transcriptome wide view of all RBP interactions with all RNAs. We benchmarked ePRINT against gold-standard CLIP technologies, then demonstrated an application by applying ePRINT to our MN differentiation protocol. We identified RBPs involved in the cell fate transition between motor neuron progenitor (MNP) and post-mitotic MN. Importantly, we uncovered RBPs that had changes in activity, but not in RNA expression, meaning that ePRINT has the potential to uncover RBPs that contribute to ALS pathology, but have been missed by RNA analysis.
Intervention or correction of dysregulated RNA regulators, such as miRs and RBPs discussed in this thesis, offer promising therapeutic targets for future ALS treatments.