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Convergent molecular mechanisms of autism resulting from mis-processing of RNA-binding proteins


About This PhD Project

Project Description

Project Code: 2020-SIDB-07

Autism has a wide variety of potential genetic causes. However, afflicted individuals share common gene expression signatures to suggest converging mechanisms and disease drivers are involved. Of relevance here, a highly coordinated and neuronal-specific programme of RNA processing events is consistently disrupted in autism that impacts multiple genes1. This includes a collection of RNA-binding proteins (RBPs) with transcriptome-wide roles as master regulators of RNA processing and translation. Perturbed processing of one such RBP, CPEB4, was revealed as a molecular mechanism underlying part of the common gene expression signature seen in autism causing detrimental neurodevelopment2. However, it remains unclear whether CPEB4 is unique as an autism master regulator.

This PhD will test the hypothesis that the mis-processing of other splicing/translation-associated RBPs in autism initiates changes to their regulatory activities that are detrimental to neurodevelopment. Initially the student will apply CRISPR/Cas9 genome-editing to modify the DNA code of three prioritised RBPs in human induced pluripotent stem cells (hiPSCs) such that they become mis-processed like in autism when expressed. The candidate will differentiate these lines into brain organoids3 using protocols established in the Mason lab. Subsequent comparisons between edited and control organoids using standard molecular biology techniques (e.g. western blotting, immunohistochemistry) and RNA sequencing will characterise the functional impact of the changed RBP activity on neurodevelopment. The candidate will next use the iCLIP method to determine interactions of each RBP with their RNA targets in a transcriptome-wide manner4. This positional information of RBP binding will be related to the RNA processing/translation defects detected after RBP mis-processing to guide final validation experiments that mechanistically explain the disruption of key targets underlying observed neurodevelopmental phenotypes.

Taken together, the student will learn state-of-the-art methods to determine how autism-relevant changes to specific regulatory RBPs contribute to detrimental neurodevelopmental phenotypes. Notably, as the studied RBP misprocessing events are observed across idiopathic autism cases with no known cause, the project is expected to identify novel converging mechanisms underlying autism and identify master regulators of autism for future therapeutic intervention.

References

1. Irimia M et al. A highly conserved program of neuronal microexons is misregulated in autistic brains. Cell. 2014 Dec 18;159(7):1511-23.

2. Parras A et al. Autism-like phenotype and risk gene mRNA deadenylation by CPEB4 mis-splicing. Nature. 2018 Aug;560(7719):441-446.

3. Marshall et al. Mouse vs. Man: Organoid models of brain development and disease. Brain Research. 2019 Dec 1; 1724:146427

4. Sibley CR. Individual Nucleotide Resolution UV Cross-Linking and Immunoprecipitation (iCLIP) to Determine Protein-RNA Interactions. Methods Mol Biol. 2018;1649:427-454

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