Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event.

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.

Introduction

About 40% of our genome is derived from transposable elements, such as DNA transposons and retrotransposons.[1-3] They have long been considered as junk DNAs. However, it has become clear that certain genes derived from retrotransposons play essential roles in mammalian development as newly acquired endogenous genes. We previously identified human and mouse PEG10 (Paternally expressed 10) as a novel paternally expressed imprinted gene.[4,5] PEG10 is a single-copy gene located between SGCE (Sarcoglycan epsilon) and PPP1R9A (Protein phosphatase 1, regulatory (inhibitor) subunit 9A). It is highly conserved in not only eutherian but also in marsupial mammals, but it is absent from monotreme mammals and from other vertebrates, such as birds and fish.[5,6] A structural analysis of PEG10 clearly showed that it was derived from one of the sushi groups of Ty3/Gypsy LTR (long terminal repeat) retrotransposons. PEG10 has two separate open reading frames (ORF1 and ORF2) that produce proteins similar to Gag and Pol proteins of the sushi-ichi retrotransposon from fugu fish, and still retains a -1 frame-shifting mechanism to produce Gag-Pol (ORF1 and ORF2) fusion protein, as is always seen in the Ty3/Gypsy retrotransposons and retroviruses.[4,7-9] Peg10 deficient mice, which lack both ORF1 and ORF2, have early embryonic lethality before 10.5 days post-coitus owing to severe placental defects.[10] Similarly, Peg11/Rtl1, another retrotransposon-derived imprinted gene highly conserved in the eutherian species, also plays an essential role in the maintenance of the placenta during pregnancy.[11] Its loss leads to late fetal/neonatal lethality because of placental malfunction. PEG11 retrotransposon insertion occurred before divergence of the eutherians and marsupials, but PEG11 became degenerated in the marsupial lineage.[12] Therefore, PEG11 is a eutherian-specific SIRH (Sushi-Ichi Retro-transposon Homologues) family gene, which is critical for the maintenance of the normal placental structure and function during the middle and late embryonic period in mice.[11,13]

There are 11 genes that are similar to the sushi-ichi retrotransposon (SIRH family genes, also called MART or SUSHI genes), including PEG10 and PEG11/RTL1 in humans and mice, and they are highly conserved in the eutherian mammals.[4,5,8,10,14-17] As mentioned above, PEG10/SIRH1 and PEG11/SIRH2 are paternally expressed imprinted genes on human chromosome 7q21/mouse proximal chromosome 6 and human chromosome 14q32/mouse distal chromosome 12, respectively.[13,18] In mice, Sirh3/Ldoc1l is another autosomal gene showing biallelic expression on the distal chromosome 15, while Sirh4, Sirh5, Sirh6, Sirh7, Sirh8, Sirh9, Sirh10 and Sirh11 are located on the X chromosome. To elucidate functions of all the SIRH family genes, systematic production of KO (knock out) mice for all the SIRH family genes are now in the process. It is possible that the other SIRH family genes have essential functions like PEG10/SIRH1 and PEG11/SIRH2.

With the recent availability of marsupial genome data, it is possible to screen marsupial-specific SIRH family genes by comparing them with those of eutherians and those of other vertebrate genomes. Here we report the identification of SIRH12 as a novel retrotransposon-derived gene in tammar wallaby.

Materials and Methods

Animals and tissue collection

Tammar wallabies of Kangaroo Island origin were maintained in the University of Melbourne marsupial breeding colony in grassy, outdoor enclosures. Lucerne cubes, grass and water were provided ad libitum and supplemented with fresh vegetables. Fetuses and yolk sac placentas were collected between days 23 and 25 of the 26.5-d gestation.[19,20] A series of tissues were collected from pouch young [d152 post partum (pp)]. Experimental procedures conformed to Australian National Health and Medical Research Council (1990) guidelines and were approved by the Animal Experimentation Ethics Committees of the University of Melbourne.

RT-PCR and 5′- and 3′-RACE

Genomic DNA and total RNA were prepared from fetuses and yolk sac placentas from wallaby conceptuses and several tissues from a pouch young, using TRISOL (Invitrogen), as described in manufacturer's protocol. cDNA was synthesized from 1 μg of total RNA using Superscript III reverse transcriptase (Invitrogen) with an oligo dT primer. Gene expression profiles were deduced by agarose gel electrophoresis of RT-PCR products with ethidium bromide (EtBr) staining. The primers used for the expression profiles were as follows: SIRH12-F (5′-TTTCTCCAGCTGTTCTGGCT-3′), SIRH12-R (5′-CAGGGTAGAGGGGAGGTTTC-3′), GAPDH-F (5′- AGAAAGTGGTGAAGCAGGCAT-3′) and GAPDH-R (5′-TGGAGGACATGTAGACCATGAG-3′). RACE reactions were performed with wallaby liver and large intestine using RNA SMARTER RACE cDNA Amplification kit (Clontech) according to the manufacturer's recommendations. The 5′- and 3′-RACE fragments were generated with the gene-specific primers SIRH12-5′-RACE (5′-TCCATGTGGCCAAGTTCTGAGGATTC-3′) and SIRH12-3′-RACE (5′-GAATCCTCAGAACTTGGCCACATGGA-3′), respectively. PCR conditions were as described previously.[5]

Comparative genomics analysis

Identification of SIRH family genes was performed using the TBLASTN and BLSTP program from NCBI server (http://www.ncbi.nlm.nih.gov/BLAST/) against eutherian and marsupial genomes using sushi-ichi Gag protein as a query (GenBank ID. AF030881). Sequence analysis was performed using the ClustalW program (http://clustalw.ddbj.nig.ac.jp/top-j.html). The sequences of opossum SIRH12 syntenic region [6993247–8893247 Monodelphis domestica chromosome 3 genomic contig, reference assembly (based on MonDom5)], tammar wallaby Macropus eugenii SIRH12 region, mouse Sirh12 syntenic region (5099338–6288585 Mus musculus strain C57BL/6J chromosome 13 genomic contig, MGSCv37), human SIRH12 syntenic region (c24594359–23494359 Homo sapiens chromosome 5 genomic contig, GRCh37.p2 reference primary assembly), platypus (12010399–12780399 Ornithorhynchus anatinus chromosome 1 genomic contig, reference assembly [based on Ornithorhynchus_anatinus-5.0.1)], chicken (930001–1330000 Gallus gallus chromosome Z genomic contig, reference assembly [based on Gallus_gallus-2.1)], Frog (c1800000–1200001 Xenopus (Silurana) tropicalis unplaced genomic scaffold, v4.2 XENTRscaffold_113) and Fugu (FUGU4:scaffold_49:800001:930000:-1) were extracted from NCBI (http://www.ncbi.nlm.nih.gov/). The tammar BAC (Bacterial artificial chromosome) library (MEB1) were screened using tammar SIRH12 sequence as a probe by PCR at the RIKEN Yokohama Institute. Sequence of BAC clone (MEB1-141D12), which includes SIRH12 was determined at the National Institute of Genetics by a combined shotgun/nested deletion strategy adopted to sequence the BAC inserts as described previously.[21-24] The primer sequences and conditions for their use are available on request. The sequence data have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/genbank/) under GenBank ID. JF720345. RepeatMasker (http://www.repeatmasker.org) was used for the detection of long interspersed nuclear elements (LINEs), short interspersed nuclear elements (SINEs) and LTR elements in the genomic region. Alignments were obtained using the VISTA Web server (http://www-gsd.lbl.gov/VISTA). SIRH12 syntenic regions of several species identified above were aligned using the default setting (>70% identity and >100 bp in length) of mVISTA with the LAGAN program.

Results

Identification of a SIRH family gene in marsupials

Sushi-ichi is the first full-length vertebrate LTR retrotransposon isolated from Fugu fish. The sushi-ichi Gag protein has a unique amino acid sequence among the LTR retrotransposons, so sushi-ichi retrotransposon homologues (SIRH) were screened by TBLASTN and BLASTP analyses. In humans and mice, 11 SIRH family genes have been identified previously.[4,5,8,10,14-17] (Table 1A, B). In two marsupials (the grey short-tailed opossum and tammar wallaby), five sequences were identified in tammar wallaby (Table 1C) and none in the grey short-tailed opossum. One corresponded to tammar PEG10 as previously reported.[6] Another sequence (GenBank ID. ABQO010016898) also shared high homology in the amino acid sequence with other SIRH family genes, therefore, we named it SIRH12, as a novel candidate in the SIRH gene family (Fig. 1). The remaining three sequences seemed to be non-functional because their open reading frames had accumulated multiple nonsense mutations.

Table 1.

List of SIRH family gene candidates

Gene name	SIRH number	Other alias	Accession number	Expect	Identity
A
PEG10	SIRH1	EDR, HB-1, KIAA1051, MEF3L, Mar2, Mart2, RGAG3	NM_001040152	3.00E−24	97/359 (28)
RTL1	SIRH2	MART1, Mar1, PEG11	NM_106713	3.00E−18	63/198 (32)
LDOC1L	SIRH3	DKFZp761O17121, Mar6, Mart6, dJ1033E15.2	NM_032287	7.00E−04	28/88 (32)
FAM127C	SIRH4	RP4-809E13.1, CXX1c, FLJ25577, MAR8B	NM_001078173	0.45	26/76 (35)
FAM127A	SIRH5	CXX1, MAR8C, MART8C, MGC117411, Mar8, Mart8	NM_001078171	0.35	30/93 (33)
FAM127B	SIRH6	CXX1b, DKFZp564B147, MAR8A, MGC8471	NM_001078172	0.34	33/111 (30)
LDOC1	SIRH7	BCUR1, Mar7, Mart7	NM_012317	0.002	24/76 (32)
RGAG4	SIRH8	RP11-262D11.3, 6430402L03Rik, KIAA2001, MAR5, MART5	NM_001024455	1.00E−09	41/157 (27)
ZCCHC5	SIRH9	FLJ38865, Mar3, Mart3, ZHC5	NM_152694	3.00E−12	63/254 (25)
RGAG1	SIRH10	KIAA1318, MAR9, MART9, MGC142230	NM_020769	6.00E−07	39/137 (29)
ZCCHC16	SIRH11	FLJ46608, Mar4, Mart4	NM_001004308	1.00E−05	57/242 (24)
B
Peg10	Sirh1	AA407948, Edr, HB-1, MEF3L, Mar2, Mart2, MyEF-3	NM_001040611	2.00E−28	81/253 (33)
Rtl1	Sirh2	6430411K18Rik, Mar, Mart1, Mor1, Peg11	NM_184109	4.00E−17	56/166 (34)
Ldoc1l	Sirh3	BC058638, MGC73499, Mar6, Mart6, sushi-15E3	NM_177630	5.00E−04	51/202 (26)
CAAX box 1 homolog C	Sirh4	RP23-479D16.1, 2900027G03Rik, Mar8.1, Mart8a	NM_028375	0.003	26/76 (35)
CAAX box 1 homolog A	Sirh5	Mart8b; Mar8.2A/B; 1110012O05Rik;	NM_024170	0.003	26/76 (35)
CAAX box 1 homolog B	Sirh6	Mart8c	NM_001018063	0.003	26/76 (35)
Ldoc1	Sirh7	RP23-322K17.2, Gm366, Mar7, Mart7	NM_001018087	0.81	22/76 (29)
Rgag4	Sirh8	RP23-448C18.4, 6430402L03Rik, KIAA2001, Mar5, Mart5, mKIAA2001, sushi-XC3	NM_183318	2.00E−10	4/167 (27)
Zcchc5	Sirh9	RP23-233G6.4, D430021I08Rik, Gm375, Mar3, Mart3, ZHC5, sushi-XD	NM_199468	2.00E−09	63/258 (25)
Rgag1	Sirh10	RP23-71M18.1, Gm385, KIAA1318, Mar9, Mart9, mKIAA1318, sushi-XF2	NM_001040434	3.00E−09	42/137 (31)
Zcchc16	Sirh11	RP23-319K12.1, C230031A03Rik, Mar4, sushi-XF2b	NM_001033795	2.00E−07	60/283 (22)
C
PEG10	SIRH1		ABQO010716413	2.00E−18	88/317 (27)
Degenerated			ABQO010379794	0.008	30/95 (31)
Degenerated			ABQO010296533	0.03	40/140 (28)
Degenerated			ABQO010214722	0.39	18/60 (30)
SIRH12	SIRH12		ABQO010016898	7.00E−05	16/52 (31)

A, Human genes; B, mouse genes and C, wallaby genes. Values in parenthesis are percentages values.

Note. Analysis was performed using the TBLASTN and BLAST program from NCBI server (http://www.ncbi.nlm.nih.gov/BLAST/) against human, mice, opossum and wallaby genomes using sushi-ichi Gag protein as a query (GenBank ID: AF030881).

Figure 1.

Amino acid alignment of SIRH family genes. Alignment of the amino acid sequence of the Gag-like regions of tammar SIRH12 and PEG10, mouse Sirh family genes and Gag region of sushi-ichi retrotransposon from Fugu fish is presented. CX2CX4HX4C zinc finger motif conserved in Ty3/Gypsy type retrotransposons is indicated. Highly conserved residues are in dark blue and relatively identical residues are in light blue.

Genomic structure and expression of SIRH12 in tammar wallaby pouch young tissues

SIRH12 full-length sequence consisting of 1492 bp was determined by 5′ RACE (Rapid Amplification of cDNA Ends) and 3′-RACE (GenBank ID. JF710845). SIRH12 is an intron-less gene, as are most SIRH family genes, such as SIRH3, SIRH4, SIRH5, SIRH6, SIRH7, SIRH8 and SIRH10. SIRH12 has a candidate ORF consisting of 107 amino acids showing high similarity with the Gag protein of sushi-ichi (Fig. 2A). However, it lacks CCHC zinc finger motif, conserved in the Gag protein in retrotransposons and retoviruses, and a part corresponding with Pol protein that is retained in PEG10, PEG11/RTL1 and SIRH9. LTR sequences that are usually attached at both ends of the retrotransposons and retoviruses are not recognizable as there are no terminal redundancy sequences and no homology with existing LTR sequences within both 5 kb sequences of upstream and downstream of SIRH12. Therefore, SIRH12 may be transcribed from a promoter that was derived from host genome. However, it is also possible that a promoter on the SIRH12 LTR has degenerated during evolution but still drives SIRH12 transcription. All these data suggest that SIRH12 has already lost its retotranspositional ability.

Figure 2.

Genomic structure and expression profiles of SIRH12. (A) Genomic structure of full-length tammar SIRH12. An arrow represents the direction of SIRH12 gene. UTR (untranslated region) and ORF (open reading frame) are indicated by blue and purple boxes, respectively. The primer positions used for RT-PCR are indicated by arrowheads. (B) Expression profiles of SIRH12 in the tammar fetus and yolk sac placenta (between days 23 and 25 of pregnancy). The RT-PCR products using total RNA from the tammar fetus and yolk sac placenta are shown. Expression of tammar GAPDH (glyceraldehyde-3-phosphate dehydrogenase) for each sample is shown as a control. (C) Expression profiles of SIRH12 in several tissues of wallaby pouch young (d152 pp). The RT-PCR products using total RNA from several tissues of wallaby pouch young are shown. Expression of tammar GAPDH for each sample is shown as a control.

Expression of SIRH12 was analysed in wallaby fetuses and yolk sac placentas (Fig. 2B), and several tissues from a day 152 wallaby pouch young (Fig. 2C). SIRH12 was not expressed in head and limb but was expressed in the bodies of the tammar fetuses and yolk sac placentas while, in pouch young tissues, SIRH12 was expressed in the endoderm-derived tissues, liver, pancreas, large intestine, spleen and stomach, but was not detected in the lung nor in the tissues primarily of mesodermal and ectodermal origin, namely kidney, heart, adrenal and bladder. This endodermal expression pattern in pouch young is unique compared with those of other SIRH family genes in the eutherian mammals, while expression in the placenta is relatively common in SIRH family genes.

Comparative analysis of SIRH12

To elucidate whether SIRH12 is an orthologue for either one of the SIRH1-11 genes in humans and mice, precise mapping of tammar wallaby SIRH12 was carried out by sequencing a wallaby BAC clone containing SIRH12. SIRH12 was located between two neighbouring genes, ENC1 (ectodermal-neural cortex) and RGNEF (Rho-guanine nucleotide exchange factor) that are conserved in vertebrates. In the BAC sequence, there are several syntenic regions, so-called evolutionary conserved regions (ECRs A–I in Fig. 3A), in the interval between ENC1 and RGNEF. ECRs B, C, D and I are conserved among all three mammalian groups including the egg-laying mammals (the monotreme platypus), while ECRs A, E, F, G and H are conserved only in therian mammals (the eutherians and marsupials). It is clear that tammar SIRH12 is located between ECRs C and D and that a SIRH12 orthologue in the South American opossum resides in the same location, although it is degenerated and does not have a long ORF corresponding to the wallaby SIRH12 (Fig. 3B). Importantly, in eutherian mammals there are no SIRH12 orthologues present between the ECRs C and D. The same is true of the platypus, chicken, frog and fish, indicating that SIRH12 retrotransposition occurred only in the marsupial lineage after their divergence from the eutherian mammals that occurred between 130 and 148 million years ago.[25,26]

Figure 3.

Mapping and comparative genomic analysis of the tammar wallaby SIRH12. (A) Mapping of the tammar wallaby BAC including SIRH12 between ENC1 and RGNEF gene. mLAGAN alignment of mouse, human, opossum, platypus, chicken, frog and fugu ENC1/RGNEF region and wallaby BAC sequence including SIRH12 are shown based on the mouse sequence using mVISTA program. Default parameters for mVISTA were used (conserved level, 70%). Conserved regions appear as peaks highlighted in pink (∼70% identity). When these regions coincide with exonic sequences of ENC1 or RGNEF, the peaks are shaded in purple. There are nine ECRs (evolutionary conserved regions) in the tammar BAC sequence (ECRs A–I; green boxes) between mice and opossum in a region orthologous to ENC1/RGNEF. Tammar wallaby and opossum SIRH12 are located between ECRs C and D, represented by red stars. (B) Conserved location of SIRH12 in marsupials but not in eutherians. mLAGAN alignment of opossum, mouse, human and platypus between ECRs B and E based on wallaby BAC sequences. The black arrow represents wallaby SIRH12 located between ECRs C and D. SIRH12 orthologous sequence in opossum was conserved but its protein-coding frame had degenerated by accumulation of nonsense mutations. There is no evidence of SIRH12 in eutherian mammals, monotremes, chicken and fugu.

Evolutions of SIRH family genes

Using published sequences, we compared the entire region between ENC1 and RGNEF among several vertebrates from fish (fugu) to mammals. As is reported, the size of this region is the smallest in fugu fish and that of opossum is the largest. There are numerous LINEs and SINEs (red and green bars in Fig. 4) in all mammalian groups. By insertions of these elements mammalian genomes become longer than those of fugu fish and chicken. Consistent with the previous report, LTR-type retrotransposons are absent in the platypus[27] (blue bars in Fig. 4). The PEG10 retrotransposon insertion occurred in the genome of the therian ancestor and was incorporated into the genomes of both marsupials and eutherians[6] (Fig. 5A), while PEG11/RTL1 is a eutherian-specific gene[12] (Fig. 5B). It is highly likely that other eutherian SIRH family genes, SIRH3 to SIRH11, are not present in the marsupials (M. Naruse, M. Ishii and R. Ono, unpublished data), suggesting that their retrotranspositions occurred only in the eutherian lineage. Our data in this report indicate that the original SIRH12 was retrotransposed into the ancestral marsupial genome after the eutherian–marsupial divergence and became incorporated into the tammar wallaby genome but degenerated in that of the opossum (Fig. 5C).

Figure 4.

Retrotransposons between ENC1 and RGNEF regions and chromosomal location of wallaby SIRH12. Red and green bars on the right side of the center lines represent LINE and SINE sequences, respectively, and blue bars on the left side represent LTR retrotransposons. Orange, purple and black bars on the left side represent ENC1, RGNEF and SIRH12, respectively. Long pink bars on the right side represent ECRs B, C and D from top to bottom. SIRH12 is located between ECRs C and D.

Figure 5.

Possible evolutionary pathway of the SIRH family genes in mammals. (A) PEG10 insertion occurred in a therian ancestor and domesticated before the split of marsupials and eutherians. (B) PEG11/RTL1 insertion occurred in a therian ancestor but domesticated only in the eutherians and collapsed in the marsupials. (C) SIRH12 insertion occurred in a marsupial ancestor and domesticated at least in wallaby but collapsed in opossum.

Discussion

In this study, we identified a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. Comparative genomic analysis suggests that SIRH12 is present in the marsupial lineage but is not present in the eutherian and monotreme lineages. Together with other SIRH genes, PEG10, PEG11/RTL1 and SIRH3-11, it is probable that the sushi-ichi-like retrotransposons were once active and retrotransposed around the time of the divergence between marsupials and eutherians, contributing to the evolution of both the extant eutherian and marsupial mammals. In general, as retrotransposons are harmful for host animals, they tend to be inactivated by DNA methylation, nonsense and frame-shift mutations.[28] However, some were incorporated into their genomes and became functional, so were selected positively, presumably because they were advantageous to their host animals.[10,11,29-33] Although it remains unknown when the opossum SIRH12 degenerated after its incorporation in the marsupial lineage, SIRH12 may be functional at least in the wallaby because its protein-coding frame has been maintained and is actively transcribed in several tissues. As species-specific genes are strong candidates for species-specific functions, it would be interesting to know whether SIRH12 has a specific function. The opossum and the tammar are very different marsupials. The tammar is a macropodid marsupial of the highly evolved kangaroo family, but the grey short-tailed opossum is a generalized marsupial closest in form to the ancestral marsupials. The tammar placenta is more highly specialized than that of the opossum[34] and there are many structural and functional differences between tammar and opossum placentas.[35-37] Although both Peg10 and Peg11 are derived from the sushi-ichi retrotransposon and have Gag and Pol-like sequences, they have biologically very different functions: the former has a function in the formation of spongiotrophoblast and labyrinth layer, while the latter has a function in the maintenance of feto-maternal interface.[10,11] Therefore, SIRH family genes could have a wide variety of functions not only in the placenta but in some other organs to perform eutherian- or marsupial-specific functions. Because tammar SIRH12 is expressed in the yolk sac placenta, it is therefore possible that SIRH12 fulfils some role in tammar placentation.

Funding

This work was supported in part by Grants-in-Aid for Scientific Research on Priority Area (R.O.) and Innovative Areas ‘Genome Science’ (R.O., Y.K., A.T.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, Grant-in-Aid for Young Scientist (R.O.), Creative Science Research (F.I. and T.K.-I.), and Bilateral Program on Joint Research Project (F.I.) from the Japan Society for the Promotion of Science (J.S.P.S.), grants from Mochida Memorial Foundation for Medical and Pharmaceutical Research and The Nakajima Foundation (R.O.). M.B.R. was supported by an Australian Research Council Federation Fellowship, and G.S. and M.B.R. by the ARC Centre of Excellence in Kangaroo Genomics.