Evolution and Diversity of Pre-mRNA Splicing in Highly Reduced Nucleomorph Genomes

GBE Evolution and Diversity of Pre-mRNA Splicing in Highly Reduced Nucleomorph Genomes Donald K. Wong1, Cameron J. Grisdale1,2, and Naomi M. Fast1,* 1 Department of Botany, University of British Columbia, Vancouver, British Columbia, Canada 2 Present address: Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada Accepted: May 30, 2018 Data deposition: Sequenced reads from this project have been deposited at NCBI’s Sequence Read Archive (SRA) under the accession SRP129823. Abstract Eukaryotic genes are interrupted by introns that are removed in a conserved process known as pre-mRNA splicing. Though wellstudied in select model organisms, we are only beginning to understand the variation and diversity of this process across the tree of eukaryotes. We explored pre-mRNA splicing and other features of transcription in nucleomorphs, the highly reduced remnant nuclei of secondary endosymbionts. Strand-specific transcriptomes were sequenced from the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans, whose plastids are derived from red and green algae, respectively. Both organisms exhibited elevated nucleomorph antisense transcription and gene expression relative to their respective nuclei, suggesting unique properties of gene regulation and transcriptional control in nucleomorphs. Marked differences in splicing were observed between the two nucleomorphs: the few introns of the G. theta nucleomorph were largely retained in mature transcripts, whereas the many short introns of the B. natans nucleomorph are spliced at typical eukaryotic levels (>90%). These differences in splicing levels could be reflecting the ancestries of the respective plastids, the different intron densities due to independent genome reduction events, or a combination of both. In addition to extending our understanding of the diversity of pre-mRNA splicing across eukaryotes, our study also indicates potential links between splicing, antisense transcription, and gene regulation in reduced genomes. Key words: RNA-Seq, transcriptome, intron retention, cryptophyte, cryptomonad, chlorarachniophyte. Introduction The regulation and flow of information within a cell are vital processes. Many genes in eukaryotes are interrupted with intervening sequences known as introns, which are removed from transcripts via a ubiquitous process known as pre-mRNA splicing. A large complex of proteins and small nuclear RNAs (snRNAs) known as the spliceosome mediates this process. The proper assembly of the spliceosome and subsequent intron removal require conserved intronic sequence signals such as the 50 splice site (most often “GU”), the 30 splice site (most often “AG”), and a biochemically important branch point adenosine residue (Will and Lührmann 2011). The presence of introns and conserved spliceosomal components across eukaryotes suggests that splicing is a mechanistically conserved process present in the last common ancestor of eukaryotes. Even organisms that have highly reduced or highly derived genomes can still have introns. Typically, these organisms have introns that are few in number, are short (30 bp or less), or are both. In organisms with intron-sparse genomes, their spliceosomes often possess a reduced set of components (Katinka et al. 2001; Grisdale et al. 2013; Stark et al. 2015), and studying such reduced systems could provide insight into the core mechanisms of splicing. Although rare, there are examples of genomes that have lost all introns (Lane et al. 2007; Cuomo et al. 2012). Although splicing has been studied extensively in budding yeast and humans, it is assumed that this process occurs with little variation across eukaryotes. However, splicing has recently been analyzed in detail in two very different organisms with reduced genomes, extending our understanding of the diversity of pre-mRNA splicing. The microsporidian intracellular parasite Encephalitozoon cuniculi has an extremely tiny genome of 2.9 Mbp, with only 37 annotated introns (Katinka et al. 2001; Lee et al. 2010). The extremophilic red ß The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an OpenAccess article distributedunder the terms ofthe Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/),whichpermits noncommercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Genome Biol. Evol. 10(6):1573–1583. doi:10.1093/gbe/evy111 Advance Access publication June 1, 2018 1573 *Corresponding author: E-mail: . GBE Wong et al. 1574 observed so far (from both cryptophytes and chlorarachniophytes) carry their tiny genomes on three short linear chromosomes (Douglas et al. 2001; Gilson et al. 2006; Lane et al. 2007; Tanifuji et al. 2011; Moore et al. 2012; Tanifuji, Onodera, Brown, et al. 2014; Suzuki et al. 2015). Whether or not there is a functional significance to this convergence, rather than a mere coincidence, remains to be seen. As with any organellar genome, very few genes remain; many have been transferred to the host nucleus or lost. The vast majority of remaining nucleomorph genes is housekeeping genes, such as chaperone proteins, ribosomal proteins and those involved in DNA replication, along with the genes for rRNAs and an incomplete set of tRNAs (Douglas et al. 2001; Gilson et al. 2006; Lane et al. 2007; Tanifuji et al. 2011; Moore et al. 2012; Tanifuji, Onodera, Brown, et al. 2014; Suzuki et al. 2015). Introns have been found in all but one of the nucleomorph genomes sequenced to date (Douglas et al. 2001; Gilson et al. 2006; Lane et al. 2007; Tanifuji et al. 2011; Moore et al. 2012; Tanifuji, Onodera, Brown, et al. 2014; Suzuki et al. 2015). However, the density of introns in the nucleomorph genomes of cryptophytes and chlorarachniophytes is quite different. For example, the cryptophyte G. theta has 485 tightly packed protein-coding genes in its 550 kbp nucleomorph genome, and only 17 of these genes are interrupted by introns that range from 42 bp to 52 bp in length (Douglas et al. 2001). In contrast, the smaller (370 kbp) nucleomorph genome of B. natans has almost 900 extremely short (18–21 bp) introns that interrupt a majority of the 283 protein-coding nucleomorph genes (Gilson et al. 2006; Tanifuji, Onodera, Brown, et al. 2014). Whereas canonical 50 and 30 splice sites are present in these tiny introns, other commonly conserved splicing motifs such as the branch donor adenosine are not discernible (Gilson et al. 2006; Tanifuji, Onodera, Brown, et al. 2014). There have been a number of studies on the peculiarities of transcription in nucleomorphs (Williams et al. 2005; Hirakawa et al. 2011; Hirakawa et al. 2014; Tanifuji, Onodera, Moore, et al. 2014; Suzuki et al. 2016; Sanita Lima and Smith 2017), although studies about the unique introns and pr (...truncated)