Lesley J. Collins

Genomics and the Irreducible Nature of Eukaryote Cells

Large-scale comparative genomics in harness with proteomics has substantiated fundamental features of eukaryote cellular evolution. The evolutionary trajectory of modern eukaryotes is distinct from that of prokaryotes. Data from many sources give no direct evidence that eukaryotes evolved by genome fusion between archaea and bacteria. Comparative genomics shows that, under certain ecological settings, sequence loss and cellular simplification are common modes of evolution. Subcellular architecture of eukaryote cells is in part a physical-chemical consequence of molecular crowding; subcellular compartmentation with specialized proteomes is required for the efficient functioning of proteins.

Use of RNA secondary structure for studying the evolution of RNase P and RNase MRP

Abstract. Secondary structure is evaluated for determining evolutionary relationships between catalytic RNA molecules that are so distantly related they are scarcely alignable. The ribonucleoproteins RNase P (P) and RNase MRP (MRP) have been suggested to be evolutionarily related because of similarities in both function and secondary structure. However, their RNA sequences cannot be aligned with any confidence, and this leads to uncertainty in any trees inferred from sequences. We report several approaches to using secondary structures for inferring evolutionary trees and emphasize quantitative tests to demonstrate that evolutionary information can be recovered. For P and MRP, three hypotheses for the relatedness are considered. The first is that MRP is derived from P in early eukaryotes. The next is that MRP is derived from P from an early endosymbiont. The third is that both P and MRP evolved in the RNA-world (and the need for MRP has since been lost in prokaryotes). Quantitative comparisons of the pRNA and mrpRNA secondary structures have found that the possibility of an organellar origin of MRP is unlikely. In addition, comparison of secondary structures support the identity of an RNase P–like sequence in the maize chloroplast genome. Overall, it is concluded that RNA secondary structure is useful for evaluating evolutionary relatedness, even with sequences that cannot be aligned with confidence.

RNase MRP and the RNA processing cascade in the eukaryotic ancestor

BackgroundWithin eukaryotes there is a complex cascade of RNA-based macromolecules that process other RNA molecules, especially mRNA, tRNA and rRNA. An example is RNase MRP processing ribosomal RNA (rRNA) in ribosome biogenesis. One hypothesis is that this complexity was present early in eukaryotic evolution; an alternative is that an initial simpler network later gained complexity by gene duplication in lineages that led to animals, fungi and plants. Recently there has been a rapid increase in support for the complexity-early theory because the vast majority of these RNA-processing reactions are found throughout eukaryotes, and thus were likely to be present in the last common ancestor of living eukaryotes, herein called the Eukaryotic Ancestor.ResultsWe present an overview of the RNA processing cascade in the Eukaryotic Ancestor and investigate in particular, RNase MRP which was previously thought to have evolved later in eukaryotes due to its apparent limited distribution in fungi and animals and plants. Recent publications, as well as our own genomic searches, find previously unknown RNase MRP RNAs, indicating that RNase MRP has a wide distribution in eukaryotes. Combining secondary structure and promoter region analysis of RNAs for RNase MRP, along with analysis of the target substrate (rRNA), allows us to discuss this distribution in the light of eukaryotic evolution.ConclusionWe conclude that RNase MRP can now be placed in the RNA-processing cascade of the Eukaryotic Ancestor, highlighting the complexity of RNA-processing in early eukaryotes. Promoter analyses of MRP-RNA suggest that regulation of the critical processes of rRNA cleavage can vary, showing that even these key cellular processes (for which we expect high conservation) show some species-specific variability. We present our consensus MRP-RNA secondary structure as a useful model for further searches.

AN APPROACH TO TRANSCRIPTOME ANALYSIS OF NON-MODEL ORGANISMS USING SHORT-READ SEQUENCES

Transcriptome analysis using high-throughput short-read sequencing technology is straightforward when the sequenced genome is the same species or extremely similar to the reference genome. We present an analysis approach for when the sequenced organism does not have an already sequenced genome that can be used for a reference, as will be the case of many non-model organisms. As proof of concept, data from Solexa sequencing of the polyploid plant Pachycladon enysii was analysed using our approach with its nearest model reference genome being the diploid plant Arabidopsis thaliana. By using a combination of mapping and de novo assembly tools we could determine duplicate genes belonging to one or other of the genome copies. Our approach demonstrates that transcriptome analysis using high-throughput short-read sequencing need not be restricted to the genomes of model organisms.

High Throughput Genome-Wide Survey of Small RNAs from the Parasitic Protists Giardia intestinalis and Trichomonas vaginalis

RNA interference (RNAi) is a set of mechanisms which regulate gene expression in eukaryotes. Key elements of RNAi are small sense and antisense RNAs from 19 to 26 nt generated from double-stranded RNAs. MicroRNAs (miRNAs) are a major type of RNAi-associated small RNAs and are found in most eukaryotes studied to date. To investigate whether small RNAs associated with RNAi appear to be present in all eukaryotic lineages, and therefore present in the ancestral eukaryote, we studied two deep-branching protozoan parasites, Giardia intestinalis and Trichomonas vaginalis. Little is known about endogenous small RNAs involved in RNAi of these organisms. Using Illumina Solexa sequencing and genome-wide analysis of small RNAs from these distantly related deep-branching eukaryotes, we identified 10 strong miRNA candidates from Giardia and 11 from Trichomonas. We also found evidence of Giardia short-interfering RNAs potentially involved in the expression of variant-specific surface proteins. In addition, eight new small nucleolar RNAs from Trichomonas are identified. Our results indicate that miRNAs are likely to be general in ancestral eukaryotes and therefore are likely to be a universal feature of eukaryotes.

Combined experimental and computational approach to identify non-protein-coding RNAs in the deep-branching eukaryote Giardia intestinalis

Non-protein-coding RNAs represent a large proportion of transcribed sequences in eukaryotes. These RNAs often function in large RNA–protein complexes, which are catalysts in various RNA-processing pathways. As RNA processing has become an increasingly important area of research, numerous non-messenger RNAs have been uncovered in all the model eukaryotic organisms. However, knowledge on RNA processing in deep-branching eukaryotes is still limited. This study focuses on the identification of non-protein-coding RNAs from the diplomonad parasite Giardia intestinalis, showing that a combined experimental and computational search strategy is a fast method of screening reduced or compact genomes. The analysis of our Giardia cDNA library has uncovered 31 novel candidates, including C/D-box and H/ACA box snoRNAs, as well as an unusual transcript of RNase P, and double-stranded RNAs. Subsequent computational analysis has revealed additional putative C/D-box snoRNAs. Our results will lead towards a future understanding of RNA metabolism in the deep-branching eukaryote Giardia, as more ncRNAs are characterized.

Mutational Dynamics of Aroid Chloroplast Genomes

A characteristic feature of eukaryote and prokaryote genomes is the co-occurrence of nucleotide substitution and insertion/deletion (indel) mutations. Although similar observations have also been made for chloroplast DNA, genome-wide associations have not been reported. We determined the chloroplast genome sequences for two morphotypes of taro (Colocasia esculenta; family Araceae) and compared these with four publicly available aroid chloroplast genomes. Here, we report the extent of genome-wide association between direct and inverted repeats, indels, and substitutions in these aroid chloroplast genomes. We suggest that alternative but not mutually exclusive hypotheses explain the mutational dynamics of chloroplast genome evolution.

Ancestral RNA: The RNA biology of the eukaryotic ancestor

Our knowledge of RNA biology within eukaryotes has exploded over the last five years. Within new research we see that some features that were once thought to be part of multicellular life have now been identified in several protist lineages. Hence, it is timely to ask which features of eukaryote RNA biology are ancestral to all eukaryotes. We focus on RNA-based regulation and epigenetic mechanisms that use small regulatory ncRNAs and long ncRNAs, to highlight some of the many questions surrounding eukaryotic ncRNA evolution.

Are you my mother? Phylogenetic analysis reveals orphan hybrid stick insect genus is part of a monophyletic New Zealand clade.

The hybrid stick insect genus Acanthoxyla Uvarov 1944 is unusual for an obligate parthenogen, in the extreme morphological diversity it exhibits that has led to eight species being recognised. The New Zealand sexual species Clitarchus hookeri [White, A. 1846. The zoology of the Voyage of H.M.S. Erebus and Terror. In: 1 Insects of New Zealand. E.W. Janson, London.] is the putative parental species in the hybridization that gave rise to the hybrid lineage Acanthoxyla. In an effort to identify the maternal ancestor of Acanthoxyla we sequenced nuclear 28S rDNA and/or mtDNA COI & COII of all nine endemic New Zealand stick insect genera, representing 17 of the 22 described species. We also sequenced 28S from eight non-New Zealand stick insects to supplement published 28S sequence data that provided a taxonomically and geographically broad sampling of the phasmids. We applied a novel search algorithm (SeqSSi=Sequence Similarity Sieve) to assist in selection of outgroup taxa for phylogenetic analysis prior to alignment. Phylogenetic reconstructions resolved an exclusively New Zealand clade to which the maternal lineage of Acanthoxyla belonged, but did not support existing higher level taxonomy of stick insects. We did not find a sexual maternal species for Acanthoxyla but phylogenetic relationships indicate that this species lived in New Zealand and could be classified among the New Zealand Phasmatinae. Among the available taxa, the nearest evolutionary neighbours to the New Zealand phasmid fauna as a whole were predominantly from the New Zealand region (Fiji, Australia, New Guinea, New Caledonia and South America). As it appears to be an orphan, it is interesting to speculate that a combination of parthenogenetic reproduction and/or hybrid vigour in Acanthoxyla may have contributed to the extinction of its mother.

An efficient greedy K-means algorithm for global gene trajectory clustering

Optimal clustering of co-regulated genes is critical for reliable inference of the underlying biological processes in gene expression analysis, for which the K-means algorithm have been widely employed for its efficiency. However, given that the solution space is large and multimodal, which is typical of gene expression data, K-means is prone to produce inconsistent and sub-optimal cluster solutions that may be unreliable and misleading for biological interpretation. This paper applies a novel global clustering method called the greedy elimination method (GEM) to alleviate these problems. GEM is simple to implement, yet very effective in improving the global optimality of the solutions. Experiments over two sets of gene expression data show that the GEM scores significantly lower clustering errors than the standard K-means and the greedy incremental method.