The role of 3' UTR variation in the molecular pathogenesis of motor neuron disease. (360G-Wellcome-103760_Z_14_Z)

3' untranslated regions (3' UTRs) play critical roles in controlling mRNA translation and stability1 by presenting sequence motifs and secondary structural elements that mediate interactions with proteins and miRNAs. Of all human cell types, brain-specific mRNA isoforms have the longest 3 UTRs2 and therefore their regulation is likely to be most complex. Recent studies demonstrated that human genetic variations in UTRs impact on gene expression to a similar degree as variations in promoter seque nces3,4. Moreover, mutations perturbing the mRNA secondary structure and sites of mRNA-miRNA interactions can lead to diseases such as cancer and neurodegeneration1,4,5. These findings suggest that sequence variation in 3' UTRs is a major contributor to human disease; however, few studies have assessed the disease impact of 3' UTR variation, though this information is becoming increasingly available from re-sequencing projects. We hypothesize that 3' UTR sequence variation disrupts mRNA stab ility and translational control, thus contributing to increased protein aggregation in neurodegenerative disorders. We combine experiments in differentiated human motor neurons with computational modelling to study the impact of alternative 3' UTRs on protein biogenesis. We will identify sequence and secondary structural elements in 3' UTRs that mediate interactions with RNA-binding proteins (RBPs) and miRNAs. We will measure the functional impact of these regulatory elements on mRNA stability a nd translation. Finally, we shall examine 3' UTR mutations associated with motor neuron disease (MND; also known as amyotrophic lateral sclerosis, ALS). MND is a common but incurable disorder in which mRNA metabolism and protein homeostasis are disrupted6 (prevalence 6 per 100,000 total population). Recent re-sequencing of 16 MND-associated genes in 96 patients identified two novel mutations in the 3' UTR of the TDP-43 gene (Pittman and colleagues, unpublished observation). We shall analyse avai lable patient re-sequencing data to pinpoint mutations perturbing regulatory elements in 3' UTRs. We will validate candidate mutations using reporter assays and genome-editing technologies. Specifically, we shall apply computational and experimental genomic methods to address the following questions: 1. What is the repertoire of alternative 3' UTR usage in neuronal mRNAs? 2. How do sequence and secondary structural elements in 3' UTRs control RBP- and miRNA-binding? 3. How do 3' UT R regulatory elements influence mRNA stability and translation? 4. How do genetic variations in 3' UTRs associate with motor neuron disease (MND) via disruption of these mechanisms?