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Gene 460 (2010) 30–38

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Nucleotide sequences of B1 SINE and 4.5SI RNA support a close relationship of to blind mole (Spalacinae) and rats ()

Irina K. Gogolevskaya, Natalia A. Veniaminova 1, Dmitri A. Kramerov ⁎

Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov St., Moscow 119991, Russia article info abstract

Article history: Until recently, zokors (Myospalacinae) were assigned to the family. However, analysis of Received 13 January 2010 mitochondrial and nuclear genes suggests a sister relationship between zokors and subterranean of Received in revised form 2 April 2010 the family, namely blind mole rats (Spalacinae) and bamboo rats (Rhizomyinae). Here, we cloned Accepted 8 April 2010 and sequenced copies of the B1 short interspersed element (SINE) from the genome of Available online 22 April 2010 psilurus. The consensus nucleotide sequence of zokor B1 was very similar to spalacids and rhizomyids, but Received by N. Okada not cricetids. Similar to spalacids ( microphthalmus) and rhizomyids ( splendens), zokor contained two variants of the 4.5SI small nuclear RNA. The longer variant (L-variant, 104 nucleotides) was Keywords: found only in zokor, spalacids and rhizomyids. The short, or S-variant (98 nucleotides), had a wider Retroposon distribution; however, analysis of the nucleotide sequences of S-variants of 4.5SI RNA confirmed that zokors

Small RNA are closely related to spalacids and rhizomyids, but not to cricetids. The evolution of the 4.5SI RNA genes and 4.5S RNAI pseudogenes is discussed. Myospalax © 2010 Elsevier B.V. All rights reserved. Rodents Phylogeny

1. Introduction (2004) suggested that Myospalax is a sister group to the clade containing the Spalacinae and Rhizomyinae subfamilies.2 These discrepancies have Zokors are Asiatic burrowing rodents that comprise two genera, been attributed to the misidentification of a single sample of zokor in Myospalax and . Their taxonomic position is controversial. the study by Michaux and Catzeflis. Tullberg (1899) first suggested a relationship between zokors and two To date, phylogenetic data on zokor has been obtained primarily families of subterranean muroid rodents, Spalacidae and Rhizomyidae. through analysis of mitochondrial and nuclear genes. The possibility Later, other morphologists also assigned zokors to Spalacidae (Miller and remains that genetic similarities among zokors, mole rats and bamboo Gidley, 1918; Chaline et al., 1977). Gradually, however, an alternative rats could be the result of convergence caused by а similar classification that assigned zokors (subfamily Myospalacinae) to (subterranean) mode of life. Additionally, phylogenetic analysis of Cricetidae became accepted (Simpson, 1945; Chaline et al., 1977; gene nucleotide sequences requires complex computer programs, and Gromov and Polyakov, 1977; Carleton and Musser, 1984; Carrol, 1988; the use of different algorithms can result in different trees. Ideally, Pavlinov, 2003). Zokors could also be regarded as a separate branch gene-based phylogenetic trees should be confirmed by analysis of within arvicolines rather than an independent derivative of the ancestral other kinds of molecular markers (Shedlock and Okada, 2000; cricetid stock. Thus, Kretzoi (1955) amended arvicolines to the family Murphy et al., 2004; Kriegs et al., 2007; Kramerov and Vasetskii, level, placed myospalacines as a subfamily, and assigned the same rank 2009). We have developed several markers in our laboratory that can to true (Lemminae) and mole (Ellobiinae). This potentially shed light on the phylogeny of zokors. One of these taxonomic affiliation of zokors was recently supported by Agadjanian markers, the B1 short interspersed element (SINE) is likely not under (2009). Molecular studies initially confirmed the relationship between selection pressure (Veniaminova et al., 2007b). A second marker, the zokors and cricetids (Michaux and Catzeflis, 2000; Michaux et al., 2001). 4.5SI small nuclear RNA, has yet to be functionally defined However, the results of Norris et al. (2004) as well as Jansa and Weksler (Gogolevskaya and Kramerov, 2002). Both sequences often exhibit distinct features among families. Here, we investigated the feasibility of using these markers to determine whether zokors are a Abbreviations: SINE, short interspersed element; LINE, long interspersed element; sister group to (Cricetidae), or to mole rats (Spalacinae) and Pol III, RNA polymerase III; PAGE, polyacrylamide gel electrophoresis. bamboo rats (Rhizomyinae). ⁎ Corresponding author. Fax: +7 499 1351405. E-mail address: [email protected] (D.A. Kramerov). 1 Present address: Department of Internal Medicine, University of Michigan, 109 Zina Pitcher Place, BSRB 2468, Ann Arbor, MI 48109-2200, USA. 2 Recently, the rank of spalacids and rhizomyids has been lowered to subfamilies.

0378-1119/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2010.04.002 Author's personal copy

I.K. Gogolevskaya et al. / Gene 460 (2010) 30–38 31

Fig. 1. Alignment of nucleotide sequences of cDNA clones from golden ( auratus), brown (R. norvegicus) and Russian mole rat (S. microphthalmus)

(Gogolevskaya and Kramerov, 2002). Four variants of mole rat 4.5SI RNA are shown: S variant, major L1 variant, and minor L2 and L3 variants. Only nucleotides that differ from the hamster RNA sequence are indicated. Dashes indicate gaps. The nucleotide sequences of the PCR primers are shown above the alignment.

SINEs or short retroposons are repetitive 80 to 400 basepair (bp) hamster is very similar to rat and , differing only by a single sequences that are interspersed over the eukaryotic genome and are nucleotide (Fig. 1). On the other hand, the differences between mole amplified through reverse transcription (Kramerov and Vassetzky, rat and rat/mouse 4.5SI RNA are more considerable (eight substitu- 2005; Ohshima and Okada, 2005). The mammalian genome contains tions and two single-nucleotide insertions). 4 6 two to four families of SINEs that vary in number from 10 to 10 A long 4.5SI RNA (4.5SI RNA-L) has been uncovered in mole copies per genome. Individual nucleotide sequences are 65–90% rat that differs from the common short species (4.5SI-S) by a number similar. Most SINE families are derived from a cellular tRNA; however, of nucleotide substitutions and a 7-nucleotide insertion (Fig. 1). Here, some SINE families also descended from 7SL RNA or 5 S rRNA. SINEs we showed that the long and short 4.5SI RNA variants of mole rat are are transcribed by RNA polymerase III (Pol III) from an internal also present in bamboo rats and zokors, but not in other rodents, promoter in their 5′ region. The classical SINE promoter consists of including hamsters. These data indicate a sister relationship between two boxes (A and B) that are spaced 30–40 bp apart. SINEs are myospalacids, spalacids and rhizomyids, but not сricetids. considered nonautonomous mobile elements, since they do not encode proteins and utilize the reverse transcriptase of long 2. Materials and methods interspersed elements (LINEs) for amplification. SINEs of placental usually proliferate with the help of LINE-1. 2.1. DNA and RNA isolation and electrophoresis The first two SINE families discovered, B1 of mice, rats, and hamsters (Kramerov et al., 1979; Krayev et al., 1980; Haynes et al., 1981) and Alu Genomic DNA, ethanol-preserved tissue and live were of primates (Deininger et al., 1981), originated from 7SL RNA, a kindly provided by the researchers listed in Supplementary Table 1. component of the signal recognition particle (SRP). SRP is a cytoplasmic DNA was isolated from liver, kidney or muscle by incubation of the ribonucleoprotein present in all eukaryotes that is involved in the tissue with proteinase K followed by phenol/chloroform extraction. translation of secreted proteins (Ullu and Tschudi, 1984). SINEs derived Total RNA was isolated from the liver by the guanidine isothiocyanate from 7SL RNA have subsequently been identified in tree method (Chomczynski and Sacchi, 1987). (Scandentia), but not other placentals (Nishihara et al., 2002; Vassetzky et al., 2003; Kriegs et al., 2007). B1 SINEs are present in the genomes of 2.2. Cloning of B1 SINE all rodents analyzed to date, and B1 SINE sequence variants, typically single-nucleotide substitutions and small indels, have been identified Zokor genomic DNA (5.0 μg) was digested with EcoRI and HindIII that are specifictosomerodentfamilies(Veniaminova et al., 2007b). and then separated by 1% agarose gel electrophoresis. DNA fragments Analysis of these sequence variations (Veniaminova et al., 2007a,b; of 0.5–1.2 kb were collected by reverse electrophoresis on a DEAE Churakov et al., 2010) supports one of prevailing rodent trees. Here, we membrane. DNA was eluted from the membrane and precipitated by analyzed B1 SINE nucleotide sequences to confirm the relationship ethanol using 10 μg of glycogen as a carrier. The isolated genomic between zokors, mole rats and bamboo rats. fragments (0.5 μg) were ligated into 0.3 μg of pGEM3Z digested with

The 4.5SI small nuclear RNA was discovered in the 1970s (Ro-Choi EcoRI and HindIII and used to transform XL-1 Blue Escherichia coli cells. et al., 1972), but its function remains unknown. Synthesis of 4.5SI RNA Colony hybridization was carried out at 60 °C in 4× SSC containing 0.5% is carried out by Pol III, and it exhibits some sequence similarity to the SDS, 5× Denhardt's solution, 0.1 mg/ml boiled herring sperm DNA and 5′-region of the В2 SINE (Serdobova and Kramerov, 1998). In Mus a 32P-labeled murine B1 probe (Vassetzky et al., 2003). Nitrocellulose musculus and Rattus norvegicus, a single genomic locus harbors three filters were washed in 0.1× SSC containing 1% SDS at 42 °C and positive

4.5SI RNA genes that occupy 80 kb on chromosome 6 and 44 kb on colonies were identified by autoradiography. B1-containing E. coli chromosome 4, respectively (Gogolevskaya and Kramerov, 2010). In clones were purified by two additional rounds of colony hybridization. addition, hundreds of retropseudogenes of 4.5SI RNA are interspersed throughout the genome. 4.5SI RNA is specific to only four related 2.3. Northern hybridization rodent families: , Cricetidae, Spalacidae and Rhizomyidae 3 (Gogolevskaya and Kramerov, 2002). The 4.5SI RNA of golden RNA (30 μg) was separated by electrophoresis on 6% polyacrylamide gels containing 7 M urea. RNA was transferred onto a Hybond XL membrane using a semi-dry electroblotting apparatus (TE 77 PWR, 3 In the cited article, we followed the traditional classification considering mole rats Amersham Bioscience). Hybridization was carried out at 42 °C as and bamboo rats as two different families, Spalacidae and Rhizomyidae, respectively. described for cloning of B1 SINE, with the addition of 50% formamide to 4.5SI RNA was not studied in Rhizomyidae, but its genes/pseudogenes were found in 32 the pruinosus genome. the hybridization solution. Preparation of the P-labeled 4.5SI RNA- Author's personal copy

32 I.K. Gogolevskaya et al. / Gene 460 (2010) 30–38 rst letters of the family name as fi that coincide with those in the formal consensus are marked by dots. Dashes ; and subfamily Gerbillinae (Ger). All consensus sequences except B1_Myos are from rence from cricetid B1s (represented by subfamilies Cri-A0, Cri-A2, and Cri-B1). Eight ricidae (Hys), Thryonomyidae (Thr), Dasyproctidae (Das), Hydrochoeridae (Hyd), Caviidae vely. Other sequences are designated according to the three R. norvegicus ; Rat-A and Rat-B, M. musculus ). Alignment of B1 consensus sequences from different rodent families. The top row represents the common rodent B1 consensus sequence. The nucleotides Veniaminova et al., 2007b Fig. 2. indicate gaps. The alignment demonstrates the similarity of B1s from Spalacinae (Spal), Rhizomyinae (Rhiz), and Myospalacinae (Myos) and the diffe positions that distinguish cricetid B1s and spalacine/rhizomyine/myospalacine B1s are indicated by arrows and are shaded grey and black, respecti follows: (Dip), Zapodidae (Zap),(Cav), Anomaluridae Chinchillidae (Ano), (Chi), Pedetidae Octodontidae (Ped), (Oct), Geomyidae( and (Geo), Myocastoridae (Cas), (Myo). Ctenodactylidae Other (Cte), consensus Hyst sequences: Mus, Author's personal copy

I.K. Gogolevskaya et al. / Gene 460 (2010) 30–38 33

template and oligonucleotide primers specific for the S- and L-

variants of mole rat 4.5SI RNA (Fig. 1). cDNAs were synthesized from the 90–110 nucleotide fraction of liver RNA isolated as described for Northern hybridization. For PCR, the thermocycle parameters were as follows: 35 cycles of 95 °C for 30 s, 53 °C for 30 s, and 72 °C for 30 s. Amplified products were isolated by electrophoresis on gels contain- ing 3% NuSeave agarose (Lonza) and 1% standard agarose and then cloned into pGEM-T (Promega). All clones were verified by sequenc- Fig. 3. Northern blot analysis of rodent liver RNA using probes specific for 4.5SI RNA. The S and L variants are indicated by arrows. Mmu, M. musculus (house mouse); Mau, M. auratus ing with standard M13 primers using a BigDye Terminator sequencing (); Smi, S. microphthalmus, (Russian mole rat); Tsp, Tachyoryctes splendens kit and an ABI Prism 3100-Avant sequencer (Applied Biosystems). The (root rat); Mps, M. psilurus (zokor); Ama, Allactaga major (great jerboa). nucleotide sequences of the cloned DNA fragments were deposited in GenBank under the following accession numbers: B1 sequences, specific probe was performed as previously described (Gogolevskaya FJ184041–FJ184056; 4.5SI RNA sequences, GU066799–GU066805. and Kramerov, 2002). Multiple alignments using the ClustalW program were manually adjusted in GeneDoc. Sequence identity was determined using the 2.4. PCR, cloning, and data analysis GeneDoc program. Phylogenetic trees were constructed by the maximum parsimony method with a randomized order of sequences

For the detection of 4.5SI RNA cDNAs, genes and pseudogenes, PCR (10 times) and the bootstrap procedure (1000 replications) with the was carried out using rodent genomic DNA (10 ng) or cDNAs as the help of the MEGA4 program (Tamura et al., 2007). Dendrograms

Fig. 4. The nucleotide sequences of cloned cDNAs amplified using primers specific for the S variant (a) and L variant (b) of mole rat 4.5SI RNA. S-specific primers: SpaS.dir and SpaS. rev; L-specific primers: SpaL.dir and SpaL.rev2 (see Fig. 1). cSmi, cTsp, and cMps are cDNA clones of S. microphthalmus, T. splendens and M. psilurus, respectively. Positions with frequent nucleotide substitutions or indels are indicated by arrows and position numbers. The numbering corresponds to the full-length sequence of mole rat 4.5SI RNA (see Fig. 1).

The nucleotide sequences corresponding to the primers are not shown. Two subvariants (I and II) of the 4.5SI-L are marked by a brace. Author's personal copy

34 I.K. Gogolevskaya et al. / Gene 460 (2010) 30–38 were constructed using the Bayesian method (MrBayes 3.1, model DNA could be used to detect specific nucleotide sequences with a 7 GTR+Γ+I,10 generations without the first 17%). narrow distribution (in this case, different variants of 4.5SI RNA genes/pseudogenes) as phylogenetic markers. To this end, PCR using

3. Results primers specific for 4.5SI-S and -L RNA was performed. Amplified fragments of the expected length were detected only in S. micro-

3.1. Structure of zokor genomic B1 SINE phthalmus, T. splendens and M. psilurus (Fig. 5). The absence of 4.5SI-L RNA genes/pseudogenes in M. musculus and R. norvegicus was also A zokor (Myospalax psilurus) genomic library was screened by supported by screening of available genomic databases. hybridization with a radiolabeled probe specific for mouse B1 SINE. The amplified genomic fragments from S. microphthalmus, Seventeen randomly chosen positive clones were sequenced. B1 T. splendens and M. psilurus were cloned and sequenced. S-variant sequences were identified by alignment with the mouse B1 consensus sequences amplified from genomic DNA exhibited greater nucleotide sequence. From this multiple alignment (Supplementary Fig. 1), we sequence heterogeneity (80–100% identity) than those amplified were able to deduce a consensus sequence for M. psilurus B1. Fig. 2 from сDNAs (Fig. 6а). This higher level of heterogeneity could be due shows the consensus M. psilurus B1 sequence (B1_Myos) in comparison to the amplification of 4.5SI RNA pseudogenes. In M. psilurus, there to the B1 consensus sequences from rodents of various other families were two groups of genomic S-variant sequences: type 1 corre-

(Veniaminova et al., 2007b). Zokor B1 SINE was similar to B1 elements sponded to the 4.5SI-S RNA, whereas type 2 sequences contained nine from Spalax microphthalmus (Spalacidae) and Tachyoryctes splendens specific single-nucleotide substitutions. Type 2 sequences may (Rhizomyidae), the B1 sequences of these three rodents differing from represent non-transcribed or poorly transcribed sequences. One of the cricetid B1 subfamilies (Cri-A0, Cri-A2, and Cri-B1) by at least eight the T. splendens cDNA clones (cTsp_49_S, Fig. 4а) was a type 2 positions. Bayesian analysis of the consensus B1 sequences supported a sequence, which supported the idea that these sequences can be monophyly of Myospalax, Spalax and Tachyoryctes (Supplementary transcribed.

Fig. 2). The consensus sequences of the genomic 4.5SI-S RNAs of S. microphthalmus, T. splendens, and M. psilurus were very similar

3.2. 4.5SI RNA and zokor phylogeny to each other and to the S-variant of S. microphthalmus 4.5SI RNA (98– 100% identity), but were clearly different from hamster 4.5SI RNA at Previously, we identified two variants of 4.5SI RNA, the S- and L- three positions (marked by arrows in Fig. 7a; 92–94%). variants (98 and 104 nucleotides, respectively), in the mole rat The genomic L-variant sequences were characterized by an even S. microphthalmus (Gogolevskaya and Kramerov, 2002). The L-variant higher heterogeneity (73–100%identity)ascomparedtothe

(4.5SI RNA-L) was absent from house mouse and golden hamster. corresponding cDNAs (91–100%) and more so to S-variant sequences Northern blot analysis of liver RNA isolated from T. splendens and

M. psilurus showed that both 4.5SI-L and -S are present not only in S. microphthalmus, but in T. splendens and M. psilurus as well (Fig. 3), confirming that there is a relationship between these species.

The variants of 4.5SI RNA were further studied in T. splendens and M. psilurus using the following experiment. cDNAs were synthesized using 90–110 nt RNA fraction isolated from the liver of S. microphthalmus, T. splendens,andM. psilurus as template and oligonucleotides comple- mentary to 3′-end region of two 4.5SI RNA variants as primers. This cDNA was used in PCR with the primers specificforthe5′-ends of S and L 4.5SI RNAs. Amplified DNA fragments were cloned and then 15–17 clones per rodent species were sequenced.

Sequence analysis revealed that both the short and long 4.5SI RNA variants were present in all three species. Most of the 4.5SI-S sequences were identical (Fig. 4a), with the following exceptions: (i) clone cTsp_49_S, which contained seven single-nucleotide sub- stitutions; (ii) clone cTsp_38_S, which contained 4 additional A residues in a five-A residue block; and (iii) eight of nine clones of cSmi_N_S (S. microphthalmus), which contained a G instead of T at position 32 (98% identity).

The 4.5SI-L RNA sequences exhibited more variability than the S- variant. This variability mapped to five positions (nucleotides 19, 50, 53, 63, and 80) (Fig. 4b). Based on the identity of the nucleotides at these positions, the L-variant was divided into two subvariant classes, type I and type II. The major/minor nucleotide distributions of the five diagnostic positions was as follows (uppercase and lowercase letters represent major and minor nucleotides, respectively): subvariant I: (G/a)19, (A/g/t)50, (G/t)53, (G/a)63, and (A)80; subvariant II: (A/g)

19, C50, (A/t)53, A63, and (−)80. Of note, both types of 4.5SI-L RNA were present in all three rodent species (Fig. 4b), further supporting a sister relationship. Fig. 5. PCR analysis of rodent genomic DNA using primers specific for the S (a) and L (b) fi fi 3.3. Genomic sequences similar to 4.5SI RNA confirm the relation of variants of mole rat 4.5SI RNA. S-speci c primers: SpaS.dir and SpaS.rev; L-speci c zokors with mole and bamboo rats primers: SpaL.dir and SpaL.rev1 (see Fig. 1). Arrows indicate amplified products of the expected length. Mmu, M. musculus (house mouse); Mau, M. auratus (golden hamster); Pda, daghestanicus (Daghestan ), Eta, talpinus (mole-vole); Smi, Since RNA from exotic animals is often unavailable and can be S. microphthalmus, (Russian mole rat); Tsp, T. splendens (root rat); Mps, M. psilurus more difficult to analyze than DNA, we examined whether genomic (zokor); Ama, A. major (great jerboa). Author's personal copy

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Fig. 6. Nucleotide sequences of the cloned PCR products from genomic DNA obtained with primers specific for the S (a) and L (b) variants of mole rat 4.5SI RNA. The primers were as described for Fig. 4. gSmi, gTsp, and gMps refer to clones amplified from S. microphthalmus, T. splendens, and M. psilurus, respectively. Two types (1 and 2) of M. psilurus S variant are indicated by a curly brace. Type 1 corresponds to the S-variant gene, whereas type 2 sequences are most likely not transcribed.

(Fig. 6b). These results could reflect a high level of diversity of the L- 4. Discussion variant and/or by the presence of pseudogenes. The L-variant genomic consensus sequences of T. splendens and 4.1. B1 SINE as a molecular marker for rodent phylogenetic studies M. psilurus were much more similar to each other and to the L-variant of mole rat than to hamster 4.5SI RNA (Fig. 7b). The differences in RNA We studied B1 SINE nucleotide sequences to clarify the evolution- structure in the particular region analyzed in this study from the hamster ary relationships of zokors (Myospalacinae). The approach was based sequence included nine single-nucleotide positions (marked by arrows on the hypothesis that B1 subfamilies evolved congruently with their in Fig. 7b),aswellasa6–7 nucleotide insertion. Parsimony analysis of host species. That is, the emergence of new rodent groups was the 4.5SI RNA sequences also supported a monophyletic relationship for accompanied by the emergence of successful new B1 subfamilies that Myospalax, Spalax and Tachyoryctes (Supplementary Fig. 3). Taken evolved from previously existent subfamilies. These new subfamilies, together, the results of the current study, using both genomic DNA and often with old diagnostic traits, acquired new ones. Thus, tracing the cDNAs, clearly support a close relationship of Myospalacinae with evolution of B1 subfamilies allows one to draw conclusions about Spalacinae and Rhizomyinae, rather than with Cricetidae. rodent phylogeny. Mobile elements such as B1 sequences appear to be Author's personal copy

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Fig. 7. Comparison of the nucleotide sequences of 4.5SI RNAs of golden hamster (cMau4.5SI) and mole rat (cSmi4.5SI_S and cSmi4.5SI_L) with consensus sequences derived from cDNAs and genomic DNA fragments amplified using primers specific for the S (a) and L (b) variants. «с» and «g» refer to cDNA and genomic DNA, respectively. Smi, Tsp, and Mps are the sequences of S. microphthalmus, T. splendens, and M. psilurus, respectively. The arrows indicate the nucleotide positions that distinguish hamster 4.5SI RNA from the S variant and L1 subvariant of the mole rat.

nonfunctional, which makes them particularly good phylogenetic variant was absent only from Dipodidae. The nucleotide sequences of markers. the S-variants from Spalacidae, Rhizomyidae and Myospalacinae were Comparison of the consensus B1 sequence from M. psilurus with very similar; however, they differed considerably from those of mouse consensus sequences from other rodent families (subfamilies) and hamster (Fig. 7a). This distribution of 4.5SI RNA among rodent showed that M. psilurus В1 SINE was most similar to T. splendens species was supported by PCR analysis of genomic DNA (Fig. 5) and and S. microphthalmus (Fig. 2). There were at least three single- sequencing of cloned amplified PCR products (Figs. 6 and 7). The data nucleotide substitutions (at positions 108, 109, and 121) that were support a sister relationship between zokors and mole rats and specific to these three species. Five other positions (8/9, 70, 115, 116, bamboo rats (Norris et al., 2004; Jansa and Weksler, 2004), but not and 124) distinguished B1 of these three species from cricetids. hamsters (Simpson, 1945; Michaux and Catzeflis, 2000; Michaux Cricetidae was represented by three B1 subfamilies, two of which et al., 2001). These relationships are depicted as a tree in Fig. 8. Thus, (Cri-A0 и Cri-A2) had a unique trinucleotide insertion (CCA). None of zokors can be considered a subfamily (Myospalacinae) together with the 17 B1 sequences from M. psilurus contained this insertion. These the subfamilies of Spalacinae and Rhizomyinae in the Spalacidae results argue against a close relationship between Myospalacinae and family. The similarities in many morphological traits among zokors, Cricetidae, but confirm recent results (Norris et al., 2004; Jansa and mole rats and bamboo rats is most likely due to their common origin Weksler, 2004) supporting a relationship between Myospalacinae and rather than convergence caused by а common subterranean way of Spalacinae and Rhizomyinae. life.

4.2. The structure of 4.5SI RNA, its genes, and pseudogenes suggests a sister relationship between Myospalacinae, Spalacinae and Rhizomyinae

It has been shown previously that 4.5SI RNA genes are absent from the majority of rodent families, with the exception of Muridae, Cricetidae and Spalacidae, and most likely Rhizomyidae (Gogolevskaya and Kramerov, 2002). Due to the narrow distribution pattern of 4.5SI RNA, it is referred to as a stenoRNA (from the Greek “stenos” meaning narrow), along with other RNAs such as 4.5SH, BC1 and ВС200 (Gogolevskaya and Kramerov, 2002). Such RNAs arose relatively recently (some tens of millions years ago) and their genes were passed on from a progenitor to all descendant species. As such, these RNAs and the genes that encode them can serve as phylogenetic markers (Martignetti and Brosius, 1993; Gogolevskaya and Kramerov, 2002; Kramerov and Vasetskii, 2009). Fig. 8. The distribution of S and L variants of 4.5SI RNA among Myodonta rodents Northern blot analysis of liver RNA and sequencing of PCR- confirms the sister relationship of Spalacinae, Rhizomyinae, and Myospalacinae. “+” “−” amplified cDNAs revealed the presence of two variants of 4.5SI RNA, and denote the presence and absence of these RNAs in representatives of the families/subfamilies, respectively. The arrows indicate the putative time of emergence 4.5SI-S and 4.5SI-L, in Russian blind mole rat (Spalacidae) and root rat of the two 4.5S RNA variants. The relationships among Myodonta families have been (a member of the family Rhizomyidae), as well as in I confirmed by a number of studies (for example, Huchon et al., 2002; Steppan et al., zokor. The L-variant was absent from house mouse (Muridae), golden 2004; Veniaminova et al., 2007b). The divergence order of Spalacidae subfamilies is hamster (Cricetidae) and great jerboa (Dipodidae), whereas the S depicted in accordance with Norris et al. (2004) and Jansa and Weksler (2004). Author's personal copy

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4.3. Spalacid 4.5SI RNA diversity Appendix A. Supplementary data

The results of the current study considerably extend our knowl- Supplementary data associated with this article can be found, in edge of 4.5SI RNA and its genes/pseudogenes in rodents of the the online version, at doi:10.1016/j.gene.2010.04.002. Spalacidae family. Previously (Gogolevskaya and Kramerov, 2002), we cloned and sequenced full-length cDNAs derived from the 4.5S RNAs I References of brown rat, golden hamster and Russian mole rat. The RNAs in hamster were homogeneous, whereas in rat, RNAs were present that Agadjanian, A.K., 2009. Small mammals from the – of the Russian differed by a single nucleotide (A or U at position 48). The 4.5SI-S RNA Plain. In: Nevesskaya, L.A. (Ed.), Proceedings of the Paleontological Institute of – of mole rat was also homogeneous, but differed from brown rat 4.5S Russian Academy of Sciences, 289, pp. 629 674. I Carleton, M.D., Musser, G.G., 1984. Orders and families of recent mammals of the world. RNA at ten positions. The results of the current study showed that the In: Anderson, S., Jones, J.K. (Eds.), Muroid rodents. Wiley, New York, pp. 289–379. S-variant of 4.5SI RNA is homogeneous or near-homogeneous in root Carrol, R.L., 1988. Vertebrate paleontology and evolution. Freeman and Company, New rat and zokor (Fig. 4a). Although 2 of 25 clones were divergent, all of York. Chaline, J., Mein, P., Petter, F., 1977. Les grandes lignes d'une classification evolutive des the other clones were virtually identical, suggesting that there is a . Mammalia 41, 245–252. major S-variant type in these animals. Furthermore, the S-variant is Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid conserved among Spalacidae. In the region studied, there was a guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. single-nucleotide substitution (U to G) in mole rat as compared to the Churakov, G., Sadasivuni, M.K., Rosenbloom, K.R., Huchon, D., Brosius, J., Schmitz, J., 2010. other two species. Such evolutionary conservation implies a func- Rodent evolution: back to the root. Mol. Biol. Evol. doi:10.1093/molbev/msq019. tionally significant role for this RNA. Deininger, P.L., Jolly, D.J., Rubin, C.M., Friedmann, T., Schmid, C.W., 1981. Base sequence studies of 300 nucleotide renatured repeated human DNA clones. J. Mol. Biol. 151, We have previously characterized the L-variant of 4.5SI RNA in 17–33. mole rat (Gogolevskaya and Kramerov, 2002). Seven full-length cDNA Gogolevskaya, I.K., Kramerov, D.A., 2002. Evolutionary history of 4.5SI RNA and clones that were of identical sequence were isolated (type L1); two indication that it is functional. J. Mol. Evol. 54, 354–364. 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Biol. 1, 573–583. fl represented by a smaller number of clones (eight) than types L2 and Huchon, D., Madsen, O., Sibbald, M.J., Ament, K., Stanhope, M.J., Catze is, F., de Jong, W. W., Douzery, E.J., 2002. Rodent phylogeny and a timescale for the evolution of L3 (14 clones). L1 type cDNAs were denoted as subvariant I, while L2 Glires: evidence from an extensive taxon sampling using three nuclear genes. Mol. and L3 type sequences were denoted as subvariant II. The higher level Biol. Evol. 19, 1053–1065. of heterogeneity of the L-variant in the current study versus our Jansa, S.A., Weksler, M., 2004. Phylogeny of muroid rodents: relationships within and among major lineages as determined by IRBP gene sequences. Mol. Phylogenet. earlier work (Gogolevskaya and Kramerov, 2002) may be the result of Evol. 31, 256–276. different methods of cDNA production. For example, in the earlier Kramerov, D.A., Vasetskii, N.S., 2009. Short interspersed repetitive sequences as a work, sequences were isolated using RNA tailing, and the PCR primers phylogenetic tool. Mol. Biol. 43, 735–746. Kramerov, D.A., Vassetzky, N.S., 2005. Short retroposons in eukaryotic genomes. Int. were complementary to these synthesized tails. Rev. Cytol. 247, 165–221. The heterogeneity of the L-variant was not random. The substitu- Kramerov, D.A., Grigoryan, A.A., Ryskov, A.P., Georgiev, G.P., 1979. Long double- tions and indels were located primarily at five positions within a 67 bp stranded sequences (dsRNA-B) of nuclear pre-mRNA consist of a few highly fi abundant classes of sequences: evidence from DNA cloning experiments. Nucleic region (indicated by arrows in Fig. 4b). This nding, together with the Acids Res. 6, 697–713. observed conservation in length of the L-variant (Fig. 3), indicates that Krayev, A.S., Kramerov, D.A., Skryabin, K.G., Ryskov, A.P., Bayev, A.A., Georgiev, G.P., the RNA is under selective pressure and most likely has a functional 1980. The nucleotide sequence of the ubiquitous repetitive DNA sequence B1 role in the cell. In this case, the nucleotide sequence heterogeneity of complementary to the most abundant class of mouse fold-back RNA. Nucleic Acids Res. 8, 1201–1215. the 4.5SI-L RNA most likely does not interfere with its function, or may Kretzoi, M., 1955. Dolomys and Ondatra. Acta Geol. Hung. 3, 347–355. even expand its functionality. Kriegs, J.O., Churakov, G., Jurka, J., Brosius, J., Schmitz, J., 2007. Evolutionary history of – The number of 4.5S RNA genes in rodents of the Spalacidae family 7SL RNA-derived SINEs in supraprimates. Trends Genet. 23, 158 161. I Martignetti, J.A., Brosius, J., 1993. Neural BC1 RNA as an evolutionary marker: guinea pig is unknown. In the case of the S-variant, the number of genes seems to remains a rodent. Proc. Natl. Acad. Sci. U. S. A. 90, 9698–9702. fl fi be low. Recently, we established that there are only three 4.5SI RNA Michaux, J., Catze is, F., 2000. The bushlike radiation of muroid rodents is exempli ed genes in M. musculus and R. norvegicus, and all three genes are at the by the molecular phylogeny of the LCAT nuclear gene. Mol. Phylogenet. Evol. 17, 280–293. same genomic locus (Gogolevskaya and Kramerov, 2010). The Michaux, J., Reyes, A., Catzeflis, F., 2001. Evolutionary history of the most speciose number of genes for 4.5SI-L RNA appears to be much higher, as mammals: molecular phylogeny of muroid rodents. Mol. Biol. Evol. 18, 2017–2031. indicated by the high nucleotide sequence variability of the L-variant. Miller, G.S., Gidley, J.W., 1918. Synopsis of supergeneric groups of rodents. J. Wash. Acad. Sci. 8, 431–448. The sequences of the genomic PCR fragments (Fig. 6) suggest that Murphy, W.J., Pevzner, P.A., O'Brien, S.J., 2004. Mammalian phylogenomics comes of spalacids have pseudogenes and/or related retroelements in addition age. Trends Genet. 20, 631–639. Nishihara, H., Terai, Y., Okada, N., 2002. Characterization of Novel Alu- and tRNA- to true 4.5SI RNA genes. The sequences of these pseudogenes are Related SINEs from the tree and evolutionary implications of their origins. variable in the M. musculus and R. norvegicus genomes (Gogolevskaya Mol. Biol. Evol. 19, 1964–1972. and Kramerov, 2010), containing both nucleotide substitutions and/ Norris, R.W., Zhou, K., Zhou, C., Yang, G., William Kilpatrick, C., Honeycutt, R.L., 2004. or deletions, and they cannot be expressed. Continued analysis of 4.5SI The phylogenetic position of the zokors (Myospalacinae) and comments on the – RNA genes and pseudogenes in Spalacidae will further expand our families of muroids (Rodentia). Mol. Phylogenet. Evol. 31, 972 978. Ohshima, K., Okada, N., 2005. SINEs and LINEs: symbionts of eukaryotic genomes with a knowledge of the origin and evolution of non-protein coding RNAs. common tail. Cytogenet. Genome Res. 110, 475–490. Pavlinov, I.Y., 2003. Systematics of recent mammals. Moscow University Publisher, Acknowledgments Moscow. Ro-Choi, T.S., Reddy, R., Henning, D., Takano, T., Taylor, C., Busch, H., 1972. Nucleotide sequence of 4.5S RNA I of Novikoff hepatoma cell nuclei. J. Biol. Chem. 247, The authors are grateful to Vladimir Korablev (Institute of Soil 3205–3222. Biology, Vladivostok, Russia) for making zokor liver available. The Serdobova, I.M., Kramerov, D.A., 1998. Short retroposons of the B2 superfamily: evolution – “ ” and application for the study of rodent phylogeny. J. Mol. Evol. 46, 202 214. study was supported by the Molecular and Cell Biology program and Shedlock, A.M., Okada, N., 2000. SINE insertions: powerful tools for molecular Russian Foundation for Basic Research (grant 08-04-00350). systematics. Bioessays 22, 148–160. Author's personal copy

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