Transcriptional complexity and post-transcriptional regulation of long noncoding RNAs

Michael Clark (2012). Transcriptional complexity and post-transcriptional regulation of long noncoding RNAs PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

       
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Author Michael Clark
Thesis Title Transcriptional complexity and post-transcriptional regulation of long noncoding RNAs
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2012-01
Thesis type PhD Thesis
Supervisor Professor John Mattick
Total pages 260
Total colour pages 82
Total black and white pages 178
Language eng
Subjects 060405 Gene Expression (incl. Microarray and other genome-wide approaches)
060104 Cell Metabolism
060199 Biochemistry and Cell Biology not elsewhere classified
Abstract/Summary The mammalian genome is far more complex than originally anticipated. Recent data have shown that the vast majority is transcribed in one context or another, a phenomenon known as pervasive transcription, producing not only large numbers of mRNAs, but also large numbers of short and long RNAs with little or no protein-coding potential. High throughput transcriptomic studies have identified tens of thousands of these intergenic, antisense and intronic long noncoding RNAs (lncRNAs). While regulatory and structural functions have been ascribed to an increasing number of lncRNAs, most are unstudied and little is known about most stages in the lifecycle of lncRNAs or how they are regulated. This thesis aimed to examine the complexity of the transcriptome, and the stability, cell biology and functional annotation of lncRNAs. Despite the accumulating evidence, pervasive transcription has remained a controversial topic that has been repeatedly challenged. The first section of this thesis rebuts the most recently published claims by van Bakel et al. (2010) that transcription is not as pervasive as thought due to technical artifacts and that the level of transcription outside of many known gene regions is so low that it could be considered biological artifact. Instead, it was found that previous evidence for pervasive transcription came from multiple lines of evidence, not just the tiling arrays criticized by van Bakel et al. (2010). Reanalysis of the van Bakel et al. (2010) tiling array data also showed these performed poorly compared to the ENCODE results they were criticizing and that their sequencing of RNA transcripts was of insufficient depth and performed on too few tissues to properly detect the low level, tissue specific nature, of much pervasive transcription (including many lncRNAs). Although the transcriptome, including lncRNA expression, is being progressively characterized, little is known about the post-transcriptional regulation of lncRNAs, including their stability and decay, which must play an important role in regulating lncRNA function. In the second part of this thesis a custom microarray was used to undertake a genome-wide interrogation of the decay of lncRNAs in a mouse neuronal cell line following transcriptional blockage. The half-lives of ~850 lncRNAs and ~12,000 mRNAs were determined and contrary to expectations, most lncRNAs were not unstable, with lncRNA half-lives varying over a similar range to, (although on average less stable than), that of mRNAs. A number of lncRNA features including: intergenic genomic location, presence of introns and higher GC% correlated with increased stability. Conversely intronic genomic location and nuclear localization correlated with decreased stability. Further investigation of individual lncRNAs of known function identified Neat1 as highly unstable, demonstrating that while stability can affect function, low stability is no barrier to function. The third part of this thesis examines lncRNA localization within the cell. Mouse neuronal and myoblast cell lines were fractionated into nuclear and cytoplasmic portions and the global localization of lncRNAs analyzed using microarrays. Around 270 lncRNAs were identified as enriched in the nucleus and ~1000 in the cytoplasm demonstrating the widespread trafficking of lncRNAs and the localization of a number of known and uncharacterized transcripts. To identify the subcelluar localization of lncRNAs RNA-FISH was performed. Initially a novel method based on fragmented probes was designed and tested. This was successful in to localizing several positive control transcripts but its performance on novel candidates was inconsistent. Subsequently, multiple oligonucleotide RNA-FISH methods were performed, successfully identifying the localization of several uncharacterized lncRNAs and demonstrating the usefulness of RNA-FISH in characterizing lncRNAs. Characterization of lncRNA expression, function and now regulation is proceeding quickly, however, even known lncRNAs are generally not present or well annotated in gene databases. The final chapter describes the long noncoding RNA database (lncRNAdb), which was created to address this problem. LncRNAdb contains manual annotations for ~180 lncRNAs in eukaryotes detailing their expression, functional and structural characteristics as well as associations with other cellular components and diseases. LncRNAdb therefore provides a useful resource for the lncRNA community. In summary, the results of this thesis support the view that the genome is pervasively transcribed and that this transcriptional output includes large numbers of lncRNAs. These lncRNAs undergo complex post-transcriptional regulation as shown by the wide variation in RNA stability and common trafficking and localization of lncRNAs to various parts of the cell. These results begin to elucidate some of the most poorly understood aspects of the lncRNA lifecycle.
Keyword Long noncoding RNAs
LncRNA
RNA stability
RNA Localization
Posttranscriptional Regulation
Pervasive transcription
RNA FISH
Database
Neat1
Additional Notes Colour page numbers: 23, 28, 31, 41-45, 47, 70, 72-73, 76-77,79-85, 98, 100-101, 103, 107, 109, 112, 125-129, 131-133, 135, 137-143, 158, 160-161, 163, 165-166, 168, 170-173, 175, 177, 179, 182, 184, 186, 192-196, 207, 232-239, 247-250, 252, 259-260 A3 Landscape page number: pg103

 
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Created: Wed, 06 Jun 2012, 16:44:28 EST by Michael Clark on behalf of Library - Information Access Service