Analysis of Conservation and Chromatin Structure at Metazoan Splice Sites

Satu Nahkuri (2010). Analysis of Conservation and Chromatin Structure at Metazoan Splice Sites PhD Thesis, Institute for Molecular Bioscience, The University of Queensland.

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Author Satu Nahkuri
Thesis Title Analysis of Conservation and Chromatin Structure at Metazoan Splice Sites
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
Publication date 2010-03
Thesis type PhD Thesis
Supervisor John Mattick
Total pages 289
Total colour pages 49
Total black and white pages 240
Subjects 06 Biological Sciences
Abstract/Summary The regulation of alternative splicing remains a mystery, as introns often harbour numerous decoy motifs similar to the consensus splice site sequence, and most auxiliary splicing enhancing and inhibiting sequences targeted by splicing factors are poorly conserved. Conversely, the conservation level of exonic and intronic sequences flanking alternative splice sites is significantly higher than that around constitutive splice sites. One such highly conserved sequence located in the mammalian serotonin receptor gene is likely targeted by the HBII-52 small nucleolar RNA (snoRNA) and implicated in the regulation of alternative splicing. Given the enormous size of the non-coding transcriptome, the possibility that many non-spliceosomal non-coding RNAs contribute to the outcome of alternative splicing by hybridizing the precursor messenger RNA (pre-mRNA) was considered in this thesis. Splicing, transcription and chromatin are intricately coupled, as demonstrated by the observations that various histone modifications exert a profound effect on transcription and alternative splicing, that dozens of crucial chromatin associated factors double as splicing or transcription factors, and that nucleosomes associated with genomic DNA at transcription start and termination sites can occlude target motifs from regulatory proteins. For example, the precise positioning of the +1 nucleosome at the transcription start site is implicated in the modulation of transcription initiation, while the backtracking of elongating RNA polymerase II (RNAPII) upon encountering this nucleosome is suggested to prompt the creation of transcription initiation RNAs (tiRNAs) via 5’ cleavage of the newly transcribed pre-mRNA. In this thesis, two parallel lines of research were pursued to investigate how alternative splicing and splicing in general are regulated in animals. Firstly, sequences flanking mammalian and insect splice sites were analysed in search for highly conserved blocks that could serve as potential landing pads for cis- or trans-acting regulatory non-coding RNAs. The analysis revealed two putative mutually exclusive cis-acting RNA structures in the homothorax pre-mRNA of Drosophila melanogaster, and thousands of conserved intronic blocks near splice sites (CIBSs) in mammals. Intriguingly, the human genes harbouring CIBSs were associated with chromatin modification and maintenance. The mammalian conservation study also led to the discovery of an unannotated snoRNA gene, SNORD119, which resides in the intron of a core splicing factor (SNRPB, SmB) and likely guides the methylation of ribosomal RNA. Evolutionary analysis revealed a link between SNORD119 and the HBII-52 snoRNA, suggesting that the duplication of the protein-coding host gene had given rise to an independently evolving copy of SNORD119, which gained a new function as a splicing regulator after the divergence of marsupials from the mammalian lineage and is currently known as HBII-52. The second line of study was an analysis of nucleosome positioning with respect to splice sites. For this purpose, whole genome nucleosome density libraries derived from human and medaka ChIP-Seq studies were bioinformatically reassessed. Contrary to the prevailing assumption that the location of nucleosomes with respect to the underlying gene structure elsewhere than at transcription start and termination sites is random, this analysis showed that nucleosomes in the body of genes are preferentially positioned at vertebrate exons in somatic and sperm cells. The observed elevated average nucleosome occupancy at internal exons is independent of the modification status of the nucleosome, the GC content of the exon, and the expression level of the gene. Importantly, the finding indicates that the location of exons is recorded in the epigenetic structure of the genome and can be inherited, while the nucleotide bias observed at synonymous substitutions and codon usage may be driven by factors affecting DNA curvature and potential for nucleosomal association. It also creates a putative link between regulatory RNAs and the epigenetic control of splicing patterns, since there is increasing evidence that regulatory RNAs guide chromatin-modifying enzymes to their sites of action. This thesis also discusses a novel class of small nuclear non-coding RNAs, termed spliRNAs, that terminate exactly at the 3’ ends of spliced exons in sense with the pre-mRNA in a wide variety of animals, tissues and cell lines, and presents evidence that suggests that spliRNAs are derived from many, if not most, splice sites. As spliRNAs and tiRNAs feature similar length distribution, cellular location and expression profile, it is proposed that they have a common biogenesis. In this model, RNAPII elongation is retarded by a strictly positioned exonic nucleosome, possibly containing histone H3 trimethylated at lysine 36 (H3K36me3), to generate spliRNAs, and by the +1 nucleosome to generate tiRNAs. Taken together, these findings support the notion that epigenetic regulation operates at an exonic level rather than only at a genic level. Future studies will show whether spliRNAs serve as vectors for epigenetic information.
Keyword Chromatin
Alternative Splicing
Comparative genomics
non-coding RNAs
Additional Notes 49, 63, 72, 88, 89, 92, 94, 95, 114, 118, 123, 129, 131, 132, 136, 137, 143, 145, 147, 156, 246, 248, 252, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279

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Created: Mon, 22 Nov 2010, 05:02:01 EST by Mrs Satu Nahkuri on behalf of Library - Information Access Service