Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9698-9702, October 1993 Evolution Neural BC1 RNA as an evolutionary marker: Guinea pig remains a rodent (non-messenger-RNA/recruited retroposon/exaptation/rodent specific/rodent monophyly) JOHN A. MARTIGNETTI AND JURGEN BRosIus* Fishberg Research Center for Neurobiology, Box 1065, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029 Communicated by Stephen Jay Gould, July 21, 1993 ABSTRACT The traditional morphologicaily grounded treme proposal must also be reevaluated. For example, placement of South American guinea pig-like rodents (Cavio- Allard et al. (10) have shown that using the same method of morpha) within one of the two rodent suborders, Hystricog- comparative sequence analysis as Graur et al. (7)-maximum nathi, has been disputed by recent analysis of protein and parsimony-but dependent upon the use of an outgroup, nucleic acid sequence data. The Caviomorpha and possibly all either monophyly or polyphyly can be supported. Reexam- Hystricognathi would be considered a separate order, distinct ination ofthe data by Hasegawa et al. (11) using the maximum from the other rodent suborder, Sciurognathi, and thus of the likelihood method also does not support rodent polyphyly. order Rodentia, and would be placed closer phylogenetically to These contrasting studies highlight, as has been previously A. W.-H. noted (12), that caution is required in inferring phylogenies other mammals [Graur, D., Hide, W. & Li, (1991) strictly from the comparison ofamino acid and protein coding Nature (London) 351, 649-652]. To address the discrepancy nucleic acid sequences. between morphological comparisons and sequence analyses, we Alternative methods of molecular analyses may aid in have applied an alternative form of molecular analysis. We reaching a consensus on phylogenetic placement where mor- demonstrate that BC1 RNA, a neural-specific small cytoplas- phology and standard DNA and amino acid sequence analysis mic RNA that is the product of a retropositionally generated cannot. The presence of repetitive sequences [such as Alu gene (a gene derived by reverse transcription of RNA followed elements and tRNA-derived retroposons (13-18), pseudo- by insertion of the DNA copy into the genome), is present in genes (19, 20), and internal gene duplications (21, 22)], newly Sciurognathi and guinea pig but not in other mammalian arisen genes (23), the differential expression patterns of orders including Lagomorpha, Artiodactyla, and Primates. preexisting (24-26) or newly arisen genes, and gene loss or The species:confined, tissue-specific expression of a retroposed inactivation (27, 28) would provide "time-landmarks of ev- sequence therefore supports the morphological evidence for olution" (29) and would constitute molecular genetic mark- monophyly of Rodentia inclusive of guinea pig and demon- ers. Such characters may prove particularly useful in cases strates the usefulness of such molecular genetic markers. where radiations have occurred relatively fast with lineages Furthermore, the conservation and tissue-specific expression of connected by rather short internodes. the BC1 RNA gene in the two divergent rodent suborders BC1 RNA (30) is encoded by a tRNA-derived retroposon suggests that this macromolecule has been exapted into a such that the 5' domain of the encoded RNA shares 80% functional role (i.e., coopted into a variant or novel function) sequence similarity with tRNAAIa.t This neural-specific small in the rodent nervous system. cytoplasmic RNA (33) is complexed with proteins to form a ribonucleoprotein particle (34-36) and is located in the so- mata and dendrites of a specific subset of neurons (37). BC1 Based on a large body of morphological evidence, the order RNA has been detected in several sciurognathid rodents Rodentia, containing approximately half of all mammalian other than rats, and its brain-specific expression pattern has species (1), has been subdivided into two suborders (2). The been shown to be conserved (38). We examined whether BC1 Sciurognathi include true rats, mice, hamsters, and squirrel- RNA was present and maintained its neural-specific expres- like rodents, while the Hystricognathi, also representing a sion pattern in different mammalian orders, including guinea group of Old and New World rodent families, are now pig, as well as determined its cDNA sequences in several primarily restricted to Africa and South America. Examples rodentst to provide molecular evidence for rodent evolution- of this diverse suborder include guinea pig, chinchilla, and ary relationships. porcupine. However, challenging this morphologically estab- lished phylogenetic scheme of Rodentia is an apparent "mo- lecular paradox"-namely, most guinea pig protein and METHODS nucleic acid sequences exhibit an unusually high level of Animals. Tissue from the following animals for RNA dissimilarity when compared with their rodent orthologues isolation was used: guinea pig (Cavia porcellus; Hartley (refs. 3-5, but also see ref. 6). A recently proposed solution (7, 8) would define Cavio- *To whom reprint requests should be addressed. morpha and possibly the entire suborder Hystricognathi as a tThe BC1 RNA gene was generated by duplication of tRNAAla (31). separate mammalian order distinct from Rodentia. Unfortu- Most likely, at least one ofthe steps involved retroposition (an event nately, the proposal of a separate order addresses the mo- where RNA is reverse-transcribed into a DNA copy followed by insertion into the genome), a notion supported by the hallmark lecular data but leads to incongruence with paleontological A-rich region (32) located at the 3' end of the putative founder information and anatomical data (9). Thus, well-established tRNAAIa domain. In the new genomic environment, transcription of morphological synapomorphies between Sciurognathi and the modified tRNA ends at an RNA polymerase III transcription Hystricognathi would be reinterpreted as homoplasies (10). termination site further downstream, accounting for the greater Furthermore, the molecular evidence underlying this ex- length ofBC1 RNA (i.e., the internalization ofthe A-rich region and the acquired 3' unique domain; see Fig. 2). :The cDNA sequences reported in this paper have been deposited in The publication costs of this article were defrayed in part by page charge the GenBank data base (accession numbers U01304, U01309, and payment. This article must therefore be hereby marked "advertisement" U01310 for guinea pig, Syrian Golden hamster, and mouse, respec- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tively). 9698 Downloaded by guest on September 27, 2021 Evolution: Martignetti and Brosius Proc. Natl. Acad. Sci. USA 90 (1993) 9699 strain, male), Syrian Golden hamster (Microcricetus aureus; ofBiomathematics, Mount Sinai School ofMedicine; in some female), mouse (Mus musculus; C57BL/6 strain, female), rat instances, the sequences were further aligned manually. (Rattus norvegicus; Sprague-Dawley strain, male), and rab- bit (Oryctolagus cuniculus; New Zealand White Strain, female). Cow (Bos taurus) brain RNA was a gift from T. RESULTS AND DISCUSSION Kirchausen (Harvard University). BC1 RNA Expression Is Rodent Specific. To establish RNA Blots. Isolation of RNA, electrophoresis (20 Ag of whether BC1 RNA is present in mammalian species other total RNA per lane) on 1.2% agarose gels containing form- than rodents, an oligonucleotide probe was designed com- aldehyde, blotting to GeneScreen nylon membrane (NEN), plementary to the 3'-terminal 60 nucleotides of rat BC1 immobilization by UV-irradiation (39), and hybridization RNA and then was used to screen RNA blots under very- (without formamide) have been carried out by standard low-stringency conditions (Fig. lA). No RNA of a similar techniques (40). The amount and integrity ofthe loaded RNA size to rat BC1 RNA was detectable in rabbit or bovine were determined by staining the gel with ethidium bromide neural tissue. A band of slightly slower electrophoretic (data not shown). An oligodeoxynucleotide, HT005 (5'- mobility was detected, however, in human brain-derived A3GGTTGTGTGTGCCAGTTACCTTGTgGGTCT5GTTA- RNA. T4GTCT5-3'), complementary to the 60 3'-most nucleotides To identify this RNA, a human brain cDNA library was in rat BC1 RNA (33) was 5'-end-labeled with 32P and used as screened by using the 3' oligonucleotide probe under similar a probe (40). The filter was washed at 42°C in 5 x SSC (40) and conditions (46). All 16 clones characterized contained exposed for 6 hr on Fuji RX x-ray film at -80°C with BC200 RNA, a primate neural-specific small cytoplasmic intensifier screen. RNA (46, 47). However, BC200 RNA is phylogenetically RNA samples (30 Mg each) from guinea pig brain, liver, and distinct from BC1 RNA and consequently does not constitute kidney were separated on a 6% acrylamide gel (19:1 acryl- a primate BC1 RNA orthologue. Isolation and sequence amide/bismethyleneacrylamide) containing 7 M urea and analysis of the BC200 RNA gene reveals that BC200 RNA were transferred to GeneScreen. An oligodeoxynucleotide, represents a transcriptionally active monomeric Alu element GU033, complementary to the 3' region of guinea pig BC1 (61), which was detected on the BC1 RNA blots in Fig. 1A RNA (5'-AAAGGTTGTTTGTGTGCGCAGTTACCTTGT- because ofthe low degree of sequence similarity between the TTG-3') was 5'-end-labeled with 32P and used as a probe for 3' ends of these RNAs (see Fig. 2 and ref. 46). Therefore, hybridization at 50°C in 20% formamide (40). The final wash BC1 RNA is not conserved in lagomorphs, artiodactyls, or was in 0.5 x SSC at 55°C, and the filter was exposed overnight primates. on Fuji RX x-ray film at -80°C with intensifier screen. BC1 RNA Is Conserved and Expressed in a Neural-Specific Construction, Isolation, and Characterization of cDNA to Manner in Guinea Pig. We next probed, under high- RodentzBCl RNA. An oligo(dC) tail was added to the 3' ends stringency conditions, RNA extracted from guinea pig (Fig. of total RNA from mouse, Syrian (Golden) hamster, and 1B). An RNA species of the appropriate size that is present guinea pig brains by the method of Devos et al.