Long ncRNAs (non-protein coding transcripts generally considered longer than 200 nucleotides to be distinguished from classes of small RNAs) are abundantly transcribed from the mammalian genome. Despite their abundance, little is known about these transcripts. Although several individual long ncRNAs have been well-characterised and ascribed important cellular functions, there remains considerable controversy as to whether long ncRNAs are, in the main, functional. Indeed, their abundance has prompted many people to argue that long ncRNAs are simply transcriptional ‘noise’ generated by spurious transcription initiation events resulting from low RNA polymerase II fidelity. This thesis demonstrates that large numbers of long ncRNAs are specifically expressed along both temporal and spatial axes of mouse development in a manner consistent with a biological function. Custom-designed microarrays were employed to analyse the expression profiles of large numbers of long ncRNAs, along with protein-coding genes, in two models of cellular differentiation; the differentiation of mouse embryonic stem (ES) cells from pluripotency to differentiation along a hemopoietic lineage; and the commitment and differentiation of neural stem cells to oligodendrocytes. The core networks that include gene expression, transcription factor binding sites and chromatin domains that regulate ES cell pluripotency and lineage specification have been the subject of considerable attention and provide a detailed context in which to analyse ncRNA expression. Of those ncRNAs examined, 945 (26% of total) ncRNAs were expressed during the differentiation of ES to embryoid body (EB), of which 174 were significantly differentially expressed. Many of these ncRNAs were transcribed from genomic locations that overlapped modified chromatin domains, and in two further studied cases directly engaged with epigenetic machinery. Similarly, 332 long ncRNAs (9% of those examined) were expressed during processes of neuronal-glial fate switching, neurogenesis and oligodendrocyte progressive differentiation and termination, of which around half were also significantly differentially expressed. Furthermore, many of these ncRNAs exhibited expression profiles that coincided with pivotal events during the commitment and differentiation of neural stem cells (NSC) to mature myelinating oligodendrocytes. Consideration of the genomic context revealed many long ncRNAs were expressed from diverse places including intergenic, intronic, and imprinted loci and may overlap with, or are transcribed antisense to, protein-coding genes with previously described roles in either ES or NSC pluripotency and differentiation. This association also extended to expression profiles, where a comparative analysis often showed complex relationships of expression between ncRNAs and associated protein coding genes, suggesting a potential role for ncRNAs in regulating the expression of associated gene loci. The complexity and specificity of the long ncRNAs expression was illustrated by analysis of the in situ hybridisation (ISH) data conducted in collaboration with the Allen Brain Atlas. Of 1328 long ncRNAs, 849 (64%) were expressed in the mouse brain, 623 (47%) of which exhibited specific expression profiles associated with distinct neuroanatomical regions, cell types, or subcellular compartments. Again, examination of their genomic context revealed long ncRNAs were often associated with protein-coding genes of neurological importance and this association often extended to include linked expression profiles in the mouse brain. The comparative analysis of protein-coding gene expression relative to associated noncoding transcription also revealed an additional level of complexity in gene structure and genomic architecture. Analysis of both microarray and ISH data show 3’UTRs can exhibit discordant expression profiles relative to their associated protein coding genes, often in a tissue- and developmentally-specific manner. Indeed, a genome-wide analysis showed that the independent expression of 3’UTR transcripts is prevalent throughout the mouse genome where they may function intrinsically as long ncRNAs during development. Together, these genome-wide analyses indicate a large proportion of long ncRNAs exhibit specific expression profiles that are inconsistent with the notion they are meaningless transcriptional noise. Taken together with numerous studies published in recent years, this thesis provides evidence to support the emergence of long ncRNAs as a major functional component of the regulatory network that underpins differentiation and development in mammals and other complex organisms.
| Identifer | oai:union.ndltd.org:ADTP/254037 |
| Creators | Timothy Mercer |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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