RNA expression in a cartilaginous fish cell line reveals ancient 3′ noncoding regions highly conserved in vertebrates

Abstract

We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3 UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3 mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology.noncoding sequence ͉ sequence conservation ͉ Squalus acanthias E fforts toward a comprehensive compilation of cell biological, physiological, and genomic information from a variety of vertebrate species are providing a rich source of comparative data representing critical points in evolutionary divergence (1). Although available information is rapidly expanding for teleosts (bony fishes; ref.2), few cell biological or genomic data are available for the most primitive jawed vertebrates, the cartilaginous fish, class Chondrichthyes, largely represented by the order Elasmobranchii (3, 4). This information would extend comparative data through Ͼ450 million years of evolution. We have derived an embryonic mesenchymal stem cell line [Squalus acanthias embryo cell line (SAE)] from the elasmobranch S. acanthias, the spiny dogfish shark (5). This cell line from a cartilaginous fish provides a means to study the cell biology, physiology, and genomics of Chondrichthyes. Recently, the National Human Genome Research Institute targeted for comprehensive genomic sequencing Leucoraja erinacia, an elasmobranch of the superorder Squaliformes, related to S. acanthias, underscoring the value of Chondrichthyes for comparative biology (6) In efforts to explore the cell biology and genomics of c...

Conserved regions of the DMD 3′ UTR regulate translation and mRNA abundance in cultured myotubes

“…These regions are similar to the previously described HCEs [48, 49]. HCEs are found across the genome in both coding and non-coding regions of genes [48-50, 71]. HCEs are associated with the 3’ UTRs of regulatory genes, and may be involved in post-transcriptional gene regulation, alternative splicing, and polyadenylation [48-51].…”

Analysis and functional annotation of expressed sequence tags from in vitro cell lines of elasmobranchs: Spiny dogfish shark (Squalus acanthias) and little skate (Leucoraja erinacea)

“…We have derived the first cell lines from cartilaginous fish, SAE and LEE-1 (Parton et al., 2007, Forest et al, 2007; Hwang et al, 2008). The successful derivation of cell lines from these animals was based on the concept that the conventional, heterologous bovine serum supplementation to culture medium should be minimized and replaced with defined peptide growth factors and other growth-promoting medium components more specifically suited to the cells to be cultured (Barnes et al, 2008).…”

“…The little skate has been targeted by the National Institutes of Health Human Genome Research Institute as a species designated for full genome sequencing (http://www.genome.gov/Pages/Research/Sequencing/SeqProposals/SharkSkateSeq.pdf; http://www.sciencedaily.com/releases/2005/03/050307100237.htm). To develop additional tools for cellular, molecular and genomic studies of cartilaginous fish, we derived cell lines from embryos of S. acanthias and L. erinacia (Parton et al, 2007; Forest et al, 2007; Kobayashi et al, 2007; Hwang et al, 2008). We refer to these as the SAE cell line from the dogfish shark and LEE-1 cell line from the little skate.…”

Stage-specific gene expression during spermatogenesis in the dogfish (Scyliorhinus canicula)

“…The first one is related to the polarized organization of its testis, which allows precise definition of the stage-specific expression patterns of genes during spermatogenesis. The second one is related to its phylogenetic position which, through comparisons with all traditional vertebrate model organisms, makes it possible to identify conserved mechanisms likely to be ancestral in jawed vertebrates (Forest et al 2007, Venkatesh et al 2007. The combination of these two characteristics and the increasing accessibility of large-scale transcriptomic tools should renew the interest of this thus far largely neglected model in order to gain insight into the evolution of spermatogenesis in gnathostomes.…”