Characterisation of neuronal and autism-associated microRNAs through systems-based analysis

Williams, Sarah (2017). Characterisation of neuronal and autism-associated microRNAs through systems-based analysis PhD Thesis, Faculty of Medicine, The University of Queensland. doi:10.14264/uql.2017.438

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Author Williams, Sarah
Thesis Title Characterisation of neuronal and autism-associated microRNAs through systems-based analysis
School, Centre or Institute Faculty of Medicine
Institution The University of Queensland
DOI 10.14264/uql.2017.438
Publication date 2017-03-27
Thesis type PhD Thesis
Supervisor Charles Claudianos
Alexandre Cristino
Total pages 200
Total colour pages 32
Total black and white pages 168
Language eng
Subjects 0604 Genetics
0601 Biochemistry and Cell Biology
Formatted abstract
MicroRNAs are important regulatory factors in brain development and function. DNA variations disrupting their function may contribute to neurodevelopmental disorders such as autism. Operating at the posttranscriptional level, microRNAs regulate broad yet controlled sets of target genes typically via interacting with target sites found in 3' untranslated regions of mRNA transcripts. Efforts to identify microRNAs contributing to autism risk have involved examining differences in their expression patterns, mutations that affect their function, and their interaction with autism-risk genes. However, there have yet to be any robustly replicated candidate microRNAs supported by these functional evidences. Knowing what genes a microRNA targets is a major challenge in the field, and represents an important first step in investigating their biological role and contribution to autism.

This thesis examines the transcriptome-wide binding and interaction networks of microRNAs that potentially contribute to dysregulation of neurological processes and development of autism spectrum disorder. Empirical support for autism-relevant microRNAs was generated using systems-level approaches integrating computational predictions and biotin-tagged microRNA:mRNA capture (pulldown) experiments quantified by RNAseq analysis. First we established a database of microRNA target site predictions using a range of existing bioinformatics tools. An analysis of the distribution of individual microRNA binding sites across untranslated regions for the entire genome yielded some surprisingly distinct peaks at microRNA-specific offsets relative to the 3' stop codon of gene transcripts. The mechanistic relevance of these potential interaction peaks is yet to be elucidated, but they may co-occur or function with other regulatory elements. The next step was to investigate regulatory mutations within exome sequence data of an Australian autism cohort generated in our laboratory. We identified putative regulatory site mutations, and showed that collectively, DNA variants in the affected cohort were significantly more associated with known autism risk genes than controls. A rare single nucleotide variation in the binding 'seed' sequence of miR-873-5p found in an autism case was evaluated by microRNA:mRNA pulldown experiments, and we observed changes in global mRNA targeting. The binding profile of wild-type miR-873-5p includes several autism risk-genes, and suggests a potential role in potassium channel signalling of neurons. We further characterised the target networks of other two seed-sharing microRNAs, miR-324-3p and miR-1913-3p, involved in neural function. Results demonstrated the potential for these two related microRNAs to occupy different regulatory niches, supporting miR-324-3p's anti-proliferative function described in the literature, and suggesting a further role for the previously-uncharacterised primate-specific miR-1913 in regulating protein-synthesis and degradation pathways.

Together, these results underline the importance of taking a systems-level view when considering microRNA function, as predicted and experimentally-determined targets provide clues to pathways or mechanisms not otherwise evident. The work establishes a workflow to take a list of variants from an autism exome-sequenced cohort, prioritise microRNA gene variants, and apply microRNA:mRNA pulldown experiments to measure not only the usual binding profile, but also the difference due to a patient-derived variant. Our microRNA-centric systems-based approaches combined computational prediction and experimental validations to identify the underlying mechanisms of microRNA regulation in fundamental neurological processes and disorders such as autism.
Keyword microRNA
microRNA targeting
Autism
Autism spectrum disorders
Systems biology
hsa-miR-1913
hsa-miR-873
hsa-miR-324
Additional Notes Colour pages: 26, 31, 40, 49, 68, 74, 75,81, 84, 94, 95, 96, 98, 99, 100, 101, 107, 108, 121, 126, 130, 132, 134, 136, 137, 138, 147, 148, 149, 150, 151, 152 Pages in landscape format: 69, 80, 82, 84, 108, 109-114, 128, 137, 139-141, 154-161

Document type: Thesis
Collections: UQ Theses (RHD) - Official
UQ Theses (RHD) - Open Access
 
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Created: Thu, 09 Mar 2017, 20:51:10 EST by Sarah Williams on behalf of Learning and Research Services (UQ Library)