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    Lost in the RNA world : non-coding RNA and the spliceosome in the eukaryotic ancestor : a thesis presented in partial fulfilment of the requirements for the degree of PhD in Bioinformatics at Massey University, Palmerston North, New Zealand
    (Massey University, 2004) Collins, Lesley Joan
    The "RNA world" refers to a time before DNA and proteins, when RNA was both the genetic storage and catalytic agent of life; it also refers to today's world where non-coding RNA (ncRNA, RNA that does not code for proteins) is central to cellular metabolism. In eukaryotes, non-coding regions (introns) are spliced out of protein-coding mRNAs by the spliceosome, a massive complex comprised of five ncRNAs and about 200 proteins. This study examines the nature of the spliceosome and other non-coding RNAs, in the last common ancestor of eukaryotes, called here the eukaryotic ancestor. By looking at the differences between ncRNAs from diverse eukaryotic lineages, it may be possible to infer aspects of the eukaryotic ancestor's RNA systems. Comparing ncRNA and ncRNA-associated proteins involves the evaluation of the available software to search newly available basal eukaryotic genomes (such as Giardia lamblia and Plasmodium falciparum). ncRNAs are not often found using sequence-similarity based software, thus specialist ncRNA-search software packages were evaluated for their use in finding ncRNAs. One such program is RNAmotif, which was further developed during this study (with the help of its principle programmer), and which proved successful in recovering ncRNAs from basal eukaryotic genomes. In a similar manner, sequence-based search techniques may also fail to recover proteins from distantly related genomes. A new protein-finding technique called "Ancestral Sequence Reconstruction" (ASR) was developed in this thesis to aid in finding proteins that have diverged greatly between distantly-related eukaryotic species. A large amount of data was collected to investigate aspects of the eukaryotic ancestor, highlighting data management issues in this post-genomic era. Two databases were created P-MRPbase and SpliceSite to manage, sequence, annotation and results data from this project. Examination of the distribution of spliceosomal components and splicing mechanisms indicate that not only was a spliceosome present in the eukaryotic ancestor, it contained many of the components found in today's eukaryotes. Splicing in the eukaryotic ancestor may have used several mechanisms and have already formed links with other cellular processes such as transcription and capping. Far from being a simple organism, the last common ancestor of living eukaryotes shows signs of the molecular complexity seen today.
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    Function and Evolution of DNA Methylation in Nasonia vitripennis
    (Public Library of Science, 2013) Wang JH; Wheeler D; Avery A; Rago A; Choi J-H; Colbourne JK; Clark AG; Warren JH
    The parasitoid wasp Nasonia vitripennis is an emerging genetic model for functional analysis of DNA methylation. Here, we characterize genome-wide methylation at a base-pair resolution, and compare these results to gene expression across five developmental stages and to methylation patterns reported in other insects. An accurate assessment of DNA methylation across the genome is accomplished using bisulfite sequencing of adult females from a highly inbred line. One-third of genes show extensive methylation over the gene body, yet methylated DNA is not found in non-coding regions and rarely in transposons. Methylated genes occur in small clusters across the genome. Methylation demarcates exon-intron boundaries, with elevated levels over exons, primarily in the 5′ regions of genes. It is also elevated near the sites of translational initiation and termination, with reduced levels in 5′ and 3′ UTRs. Methylated genes have higher median expression levels and lower expression variation across development stages than non-methylated genes. There is no difference in frequency of differential splicing between methylated and non-methylated genes, and as yet no established role for methylation in regulating alternative splicing in Nasonia. Phylogenetic comparisons indicate that many genes maintain methylation status across long evolutionary time scales. Nasonia methylated genes are more likely to be conserved in insects, but even those that are not conserved show broader expression across development than comparable non-methylated genes. Finally, examination of duplicated genes shows that those paralogs that have lost methylation in the Nasonia lineage following gene duplication evolve more rapidly, show decreased median expression levels, and increased specialization in expression across development. Methylation of Nasonia genes signals constitutive transcription across developmental stages, whereas non-methylated genes show more dynamic developmental expression patterns. We speculate that loss of methylation may result in increased developmental specialization in evolution and acquisition of methylation may lead to broader constitutive expression.