- Title
- Investigating post-transcriptional mechanisms of neuronal translational control
- Creator
- Kiltschewskij, Dylan James
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2020
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Neuronal cells rely on extensively coordinated temporospatial patterns of gene expression to support the generation of functional brain circuitry and facilitate the molecular basis of phenomena including learning, memory and cognition. The highly complex nature of neuronal cytoarchitecture, coupled with the requirement for temporally restricted profiles of gene expression, necessitates a multidimensional array of post-transcriptional regulatory systems capable of fine-tuning mRNA stability and translation with digital specificity in response to a variety of cues. Short, non-coding microRNA (miRNA) constitute one major posttranscriptional system though which neuronal mRNAs may be extensively regulated in developing and mature neurons, however the mechanistic basis of miRNA function and their interplay with other post-transcriptional regulatory systems remains poorly described in the neuronal context. To investigate post-transcriptional miRNA-mRNA dynamics in the neuronal setting, integration of high-throughput RNA sequencing techniques was employed to acquire mechanistic insights into miRNA function during neuronal depolarisation, exogenous miRNA modulation and neuronal differentiation. Characterisation of mRNA expression and ribosomal occupancy after neuronal depolarisation uncovered a profound decoupling of mRNA abundance and translation. The functionally significant miRNA were usually associated with mRNA steady state rather than mRNA translation. These findings were further exemplified by characterisation of the depolarisation-associated and brain-enriched miR-1271-5p via ribosome profiling and mRNA sequencing, which also suggested that exogenous modulation of the miRNA was predominantly associated with decreased stability of complementary target mRNAs via binding sites in the 3´ untranslated region (UTR) and coding sequence (CDS). With canonical mRNA destabilisation established as the predominant mechanism of miRNA function, the interplay between miRNA expression and mRNA poly(A) tail length was examined to determine whether miRNA-mediated deadenylation was observable on a transcriptome-wide scale during neuronal differentiation. Although miRNAs were associated with poly(A) tail length to varying degrees, this study also enabled the discovery of alternative polyadenylation events which considerably impacted the expression of miRNA binding sites for affected mRNAs. These findings collectively provide strong evidence that neuronal miRNA primarily regulate mRNA stability on a system-wide scale, and may ultimately guide future exploration of the complexity and context-specificity of miRNA post-transcriptional function in the neuronal context.
- Subject
- mRNA translation; microRNA; neuron; ribosome profiling; poly(A) tail; thesis by publication
- Identifier
- http://hdl.handle.net/1959.13/1423915
- Identifier
- uon:37994
- Rights
- Copyright 2020 Dylan James Kiltschewskij
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
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| View Details Download | ATTACHMENT01 | Thesis | 36 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 201 KB | Adobe Acrobat PDF | View Details Download |