International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(3), 52-59, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 52 Identification of a Chimeric Transcript formed by Intergenic Splicing of UbiE2 and Yghl1-4 in MouseRavi Nagganatha Suganthan, Sahi Vaidurya Pratap, Morita Eugene Hayato and Abe Shunnosuke* Laboratory of Molecular Physiology, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Ehime, JAPANAvailable online at: www.isca.in, www.isca.me Received 26th October 2013, revised 6th December 2013, accepted 4th January 2014Abstract We identified a chimeric transcript UbiE2-Yghl1-4, formed by an intergenic splicing event between UbiE2 and Yghl1-4 present in tandem position on the mouse chromosome 15F1 at a distance of ~3kb. The UbiE2 is S-adenosylmethionine-dependent methyltransferase domain containing protein encoding gene, also known as mettl7a2. The Yghl1-4 is a member of YGH1/HIG1 gene family in mouse with YGHL/HIG1 exon 3 domain. The chimeric transcript is formed with exon 1 of UbiE2 fused to exons 2-4 of Yghl1-4 using the canonical splice sites from both genes. The UbiE2-Yghl1-4 is not a product of in vitro artifacts and our results showed independent expression of the parent and the chimeric transcripts. The expression of UbiE2, Yghl1-4 and UbiE2-Yghl1-4 were found exclusive to kidney only. Further, tissue localization by Non-Radioactive In Situ Hybridization (NRISH) indicated restricted expression in the epithelium of proximal tubules and adrenal cortex. The UbiE2-Yghl1-4 gene formation was found only in mouse and not in human. The characteristics of the UbiE2-Yghl1-4 chimeric transcript being specific to mouse genome and containing both the methyl transferase domain and the YGHL/HIG1 exon 3 domain appears to have functional implications in mouse and especially in the kidney, rather than merely generating genetic diversity. Keywords: Chimeric transcript, UbiE2, Yghl1-4, YGHL/HIG1mouse, kidney. Introduction RNA processing, other than conventional splicing and polyadenylation events, includes random combination of exons producing different variants of mRNAs. The alternative splicing process is not just restricted to the single gene but include exons from other genes also1,2. The phenomena of both alternative cleavage and splicing between two different or tandem genes is known as intergenic splicing which produces chimeric transcripts, thus increasing transcript and protein diversity3-5. Two major mechanisms leading to chimeric transcript formation are (a) trans-splicing between transcripts of unrelated genes and (b) formation of read-through transcripts between neighbour genes6-8. These chimeric transcripts have also been shown to express as an entity independent of the parent transcripts. Chimeric transcripts were considered to be a rare phenomenon a decade ago, but till date numerous chimeric transcripts have been identified and reported3-5, 10-18. The yellowtail growth hormone like-1 (YGHL1) cDNA (GenBank: D85880) was obtained on screening a pituitary cDNA library of Seriola quinqueradiata (Yellowtail fish or Japanese Amberjack) using the antibody for Salmon growth hormone by our research group in 199619. Later, Gracey et al.20identified the Hypoxia-Inducible Gene 1 (HIG1) cDNA (GenBank: AF266206), in liver tissue of a euryoxic fish, Gillichthys mirabilis, shown to be induced after prolonged hypoxia. The HIG1 and the yellowtail YGHL1 were found to be homologous, thus belonging to the YGHL1/HIG1 gene family. Multiple paralogs of the YGHL1/HIG1 are cloned and characterised in various eukaryotic genomes21-24. The generation of multiple copies of these mammalian gene families has been attributed to ancient polyploidization in ancestral vertebrates25-29. There are four paralogs of YGHL1/HIG1 in mouse viz. Higd1a (9F4), Higd1b (11D), yghl1-4 (15F1) and Higd2a (13B1)19. We describe a chimeric transcript as a result of intergenic splicing in mouse involving the UbiE2 (Mettl7a2) and Yghl1-4 Higd1c). These two genes are situated ~3 kb apart from each other on mouse chromosome 15F1 in tandem position. The UbiE2 is a S-adenosylmethionine-dependent methyltransferase domain containing protein encoding gene also known as mettl7a2 with unknown function existing in vertebrates while the Yghl1-4 is a member of YGH1/HIG1 gene family in mouse with YGHL/HIG1 exon 3 domain19. The intergenic splicing product of Yghl1-4 with its flanking gene UbiE2 was found as a fusion gene through EST database searches. The chimeric gene is composed of the exon 1 from UbiE2 and the exons 2-4 from the Yghl1-4, with a continuous open reading frame from the start codon in UbiE2 and the stop codon in Yghl1-4. This chimeric transcript must putatively have a novel function in mouse as it does not exist in human. We describe the intergenic splicing event of the UbiE2 and Yghl1-4 and also the expression patterns of the individual and chimeric transcripts in the mouse. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 52-59, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 53 Material and Methods Isolation of total RNA, determination of cDNA extremities, and cDNA analysis: RNA was extracted from various organs of mouse using an RNA Extraction Kit (GE Healthcare, USA). The 5'cDNA extremity was determined by the rapid amplification of cDNA ends (RACE) method. Poly (A+) RNA was purified from total RNA (100 µg) using an mRNA Purification kit (GE Healthcare, USA). After annealing 1 µg of poly (A) RNA with 10 pmoles of a gene specific reverse primer (5'-gtggagataggcacagttgat-3') derived from the partial cDNA sequence, the first strand cDNA was synthesized by incubation with ReverTraAce reverse transcriptase (Toyobo, Japan) for 1 h at 42 °C. Then, the blunt ended second strand was synthesized by simultaneous inclusion of DNA polymerase I (9U/µl), E. coliDNA ligase (6U/µl, Takara, Japan), and T4 DNA polymerase (9U/µl) in the presence of RNase H (1U/µl) in a reaction buffer composed of 33.4 mM Tris-HCl (pH 8.5), 4.6 mM MgCl, 10 mM (NHSO, 6.6 mM 2-mercaptoethanol, 0.06 mM EDTA, 0.005 % BSA, 100 mM KCl, and 0.15 mM NAD at 16 °C for 90 min., and the double-stranded cDNA obtained (5 µl) was ligated with 12.5 pmoles of an adaptor consisting of complimentary oligo DNA, (5'-gtaatacgactcactatagggcacgcgtggtcgacggcccgggctggt-3') and (5'-accagcccggg-3'), using 4 U/µl T4 DNA ligase at 16°C overnight. T4 DNA ligase, DNA polymerase I, and RNase H were obtained from Toyobo, Japan. The ligated cDNA fragments were 5'-RACE amplified by using an adapter primer (5'-gtaatacgactcactatagggc-3') with gene specific primers in 25 µl volume reaction mixtures using XL-PCR kit (Applied Biosystems, USA), and then subcloned into the pT7blue vector. Analysis of gene expression by RT-PCR: For RT-PCR analysis, the cDNA from various mouse tissues were diluted to optimize the analysis of UbiE2, Yghl1-4, UbiE2-Yghl1-4 and actin. The primer sequences are F-5'-ATGCCCTTTCTCCTCTACTCGC-3' and R-5'-AACCAGCGTGGAGCAGACCGG-3' for UbiE2 amplification; F-5'-ACTGGGATACTGTTCCAGC-3' and R-5'-TCTGATGCTAGCACTG-3' for Yghl1-4 amplification; and F-5'-ATGCCCTTTCTCCTCTACTCGC-3' and R-5'-TCTGATGCTAGCACTG-3' for the chimeric transcript amplification. The XL-PCR kit (Applied Biosystems) was used for carrying out all RT-PCR reactions. The reaction consists of an initial denaturation for 15 s at 94 \rC, then 30 cycles of: 15s at 94 \rC, 1 min at 60 \rC, 2min at 72 \rC, and then a final extension for 10 min at 72 \rC. The PCR products were electrophoresed in 0.5% TAE composed of 20 mM Tris (pH 8.0), 9.5 mM acetic acid and 0.5 mM NaEDTA; the gel irradiated at 310 nm and photographed using a gel documentation system (Pharmacia Biotech). DNA sequencing and analysis: Subcloned DNA was isolatedand purified using a FlexiPrep kit (GE Healthcare, USA), and sequenced using a Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) and an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, USA). Non-radioactive mRNA in situ hybridization: NRISH using biotin-labeled antisense riboprobes for UbiE2, Yghl1-4 and the chimeric transcript was performed on parasaggital sections of mouse embryo (Genostaff, Japan). The gene specific biotin- labeled riboprobes were synthesized from the sequences cloned in pGEM-T vector (Promega, USA) by in vitro transcription using RNA biotin labeling mix (Roche, Germany) and Thermo T7 polymerase (Toyobo, Japan). Sections were deparaffinized and rehydrated in serial dilution of ethanol, then incubated overnight at 42 °C with the riboprobes in hybridization buffer. Thereafter, the sections were sequentially washed with 4×, 2×, and 0.2× SSC buffer at 42 °C for 1 h each. Detection was achieved by treatment with 1× PBST (Phosphate buffered Saline + 0.1 % Tween 20) for 5 min, followed by treatment with blocking buffer for 3 h. The sections were treated with the alkaline phosphatase-streptavidin conjugate overnight at 4 °C. Subsequently, the sections were washed 3 times with 1× PBST for 15 min and then washed with alkaline Tris buffer. To detect the hybridized probe, the sections were incubated with nitro blue tetrazolium (NBT; 450 mg/ml) and 5-bromo-4-chloro-3- indoyl phosphate (BCIP; 175 mg/ml) in alkaline Tris buffer at 37 °C. As internal controls, NRISH using sense riboprobes were performed. Results and Discussion Identification and expression analysis of UbiE2-Yghl1-4 intergenic splicing product: An EST sequence (Genbank: CA481201) containing sequences of UbiE2 and Yghl1-4 was found while searching the EST database for sequences related to YGHL/HIG1 in mouse genome. The NCBI blast search with the EST sequence showed high similarity with exon 1 of the UbiE2; with exons 2, 3 and partially with exon 4 of Yghl1-4. To validate the EST based search, RT-PCR was performed on cDNAs from various mouse organs, using gene specific primers designed for the UbiE2 and Yghl1-4 and the UbiE2-Yghl1-4 chimeric transcript (figure 1). Relative expression of the different transcripts was normalized using the -actin as control. Interestingly, both the parent transcripts and the chimeric transcript were amplified in a tissue specific manner, expressing abundantly in the kidney only. The sequencing of the chimeric transcript PCR product showed that it is an intergenic splicing product containing the regions of UbiE2 and Yghl1-4. Figure-1 RT-PCR amplification of the UbiE2 and Yghl1-4 and UbiE2-Yghl1-4 chimeric transcript and -actin in mouse tissues (B - Brain, H - Heart, Lu – Lung, M – Muscle, Li – Liver, K - Kidney) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 52-59, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 54 Genomic organization of UbiE2 and Yghl1-4 loci and structural analysis of UbiE2-Yghl1-4: The genomic sequence analysis showed that the UbiE2 and the Yghl1-4 are present in the mouse chromosome 15F1. UbiE2 contains two exons and the Yghl1-4 contains four exons. The Yghl1-4 is situated 2766 bp upstream of the UbiE2 and both genes are in tandem, with the same orientation. The Intergenic splicing product encompasses UbiE2 exon 1, fused to Yghl1-4 exons 2–4 using the splice donor/acceptor sites of UbiE2 and Yghl1-4 mRNAs, along with the 3' UTR and the polyadenylation site of Yghl1-4 (figure 2). The intergenic splicing event removed the exon 2 of the UbiE2 and exon 1 of the Yghl1-4 as a novel intron region. The genomic organization of the loci, together with the structure of the intergenic splicing product, were compatible with intergenic splicing between UbiE2 and Yghl1-4, giving rise to the UbiE2-Yghl1-4 transcript. The complete nucleotide sequences of the UbiE2 (Genbank: AB253944), Yghl1-4(Genbank: AB253942) and the chimeric transcript (Genbank: AB110950) were deposited in NCBI database (figure 3). Localization of UbiE2 Yghl1-4 and UbiE2-Yghl1-4 chimeric transcript in mouse: To confirm the tissue-specific expression of UbiE2, Yghl1-4 and UbiE2-Yghl1-4 chimeric transcript in the mouse, NRISH was carried out on parasaggital sections of the mouse embryo using gene specific antisense riboprobes. The UbiE2 and Yghl1-4 hybridization signal were strongly detected in the kidney, whereas the signal was weak for the chimeric transcript (figure 4A). The magnified observation showed similar levels of expression but restricted to the epithelium of the proximal tubules (figure 4B) whereas, strong expression for all the transcripts in the adrenal cortex region (figure 4C) was observed. Discussion: Mouse EST database search for sequences related to YGHL/HIG1 revealed the presence of a chimeric UbiE2- Yghl1-4 transcript. This chimeric transcript seems to be an intergenic splicing product between UbiE2 and Yghl1-4. Sequence analysis indicated that UbiE2-Yghl1-4 chimeric transcript encompass UbiE2 exons 1, including the AUG initiation codon, fused in-frame to Yghl1-4 exons 2–4. There is no evidence of such a mechanism affecting these two genes in human. UbiE2 and Yghl1-4 genes map to mouse chromosome 15F1, are located in tandem and are separated by ~3 kb. Unconventionally, chimeric transcripts are formed from experimental artefacts due to template-switching activities of the reverse transcriptase (RT) or Taq polymerase30. The formation of UbiE2-Yghl1-4 has followed splicing using canonical splice sites whereas template switching activity is independent of splice sites. There is no sequence similarity between UbiE2 and Yghl1-4 that could contribute to a homology-dependent template switching. Moreover, the chimeric transcript was identified based on an EST that was obtained in the mouse EST database. The chimeric transcript consists of both the S-adenosylmethionine-dependent methyltransferase domain from the UbiE2 and the YGHL/HIG1 exon 3 domain from the Yghl1-4, so it appears to have functional implications, rather than merely generating genetic diversity, as previously hypothesized for other hybrid RNAs10. These characteristics showed that UbiE2-Yghl1-4 is not a product of in vitro artefact, but that its expression is tightly regulated, and has a functional significance. Figure-2 Structure of the mouse UbiE2 and Yghl1-4 genes and transcripts, A- Genomic structure of the two genes. B- Structure of the normal transcripts from both genes. C- Structure of the chimeric transcript UbiE2-Yghl1-4. Untranslated exon is in red and translation start and stop are indicated International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 52-59, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 55 Figure-3 Nucleotide sequence and deduced amino acid sequence of UbiE2 (A), Yghl1-4 (B) and UbiE2-Yghl1-4 chimeric mRNA (C). The initiation codon (atg) is underlined and the stop codon (taa/tag) is indicated by an asterisk. The S-adenosylmethionine-dependent methyltransferase domain and the YGHL1/HIG1 exon 3 domain sequences are highlighted in black and grey respectively International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 52-59, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 56 Figure-4(A) Figure-4(B) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 52-59, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 57 Figure-4 Expression of UbiE2, Yghl1-4 and UbiE2-Yghl1-4 chimeric mRNA in parasagittal sections of mouse embryo hybridized with antisense or sense biotin-labeled riboprobes. The signal detection shows UbiE2, Yghl1-4 and UbiE2-Yghl1-4 chimeric mRNA expression in the kidney (A), proximal tubules (Pt) (B) and the adrenal cortex (Ac) (C) Several mechanisms by which the chimeric transcripts generate have been reported previously, such as trans-splicing2,31,32, RNA transcriptional runoff4,17, or by presence of short homologous sequences33. The UbiE2-Yghl1-4 chimeric transcript formation has followed the classical intragenic trans-splicing model using the canonical splice sites (GT-AG) which was also consistent in reported intergenic trans-splicing events11, 34-36. The intergenic splicing event of the UbiE2-Yghl1-4 has occurred between the exon 2 of UbiE2 and exon 2 of the Yghl1-4, thus creating a novel intron, spanning the region between the 5' splice site (donor) of the last intron and the 3' splice site (acceptor) of the first intron of the UbiE2 and Yghl1-4, respectively. Akiva et al.reported that the former intergenic splicing type is the most abundant type in human chimeric transcript formation and classified it as “exon (n-1) to (+2) event”. The exon (n-1) to (+2) event occurs as an intergenic splicing between the n-1 exon (one before last) of the upstream gene and the second exon (+2) of the downstream gene. It should be also noted that the UbiE2-Yghl1-4 chimeric transcript was not a result of a weak termination signal for UbiE2 transcription; because RT-PCR analysis showed that normal UbiE2 and Yghl1-4 transcripts were also expressed, meaning that normal transcription termination and intergenic splicing of the two genes co-exist. Even though, the UbiE2 and Yghl1-4 orthologs are conserved on human chromosome 12q13.12 as METTL7A and HIGD1C, with a distance of ~21.5 kb between them, there is no evidence in the human cDNA or EST databases implying the chimeric transcript. Roux et al. pointed out that for several cases of chimeric transcript formations described so far, the physical distance between the genes involved does not seem to have any influence. The genes P2RY11 and PPAN are separated by only 400 bp13, whereas the DISC1 and TRAX genes are separated by 35 kb37. Although the exact reason for the absence of the chimeric transcript in human is not known, the structure and sequence of the human HIGD1C which is different from the mouse Yghl1-4, may explain the fact to certain level. The HIGD1C lacks the non-coding exon 1 as in Yghl1-4, which may be some sort of signal for the intergenic splicing to occur. The absence of the non-coding exon 1 in human counterpart may pose a hindrance to follow the mechanism as occurring in mouse. Secondly, the canonical AG acceptor site is absent at the 5' end of coding exon 1 of the HIGD1C for the reading frame to join with the exon 1 of the human METTL7A. These may be the reasons that the reading frames of both genes in human cannot be joined as in the mouse chimeric transcript. The occurrence of the intergenic splicing event between UbiE2 and Yghl1-4 only in mouse may be due to some specific genomic requirements International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(3), 52-59, March (2014) Int. Res. J. Biological Sci. International Science Congress Association 58 and the resultant chimeric transcript may have well defined functional role. Prakash et al.38 reported the presence of 270 conjoined genes in mouse genome and also that only 0.03% of the mouse conjoined genes junction exons are found conserved in human, which indicates that many chimeric transcripts might have undergone evolutionary selective pressure to be present in the mouse genome only. Naganuma et al.14 reported a chimeric mRNA transcribed from HAI-2 and H2RSP in human but not to be found in mouse; and Roux et al. reported a bovine chimeric transcript between PPARG and TSEN2 specific to cattle but absent in human and mouse genomes. Frenkel-Morgenstern et al. reported that chimeric transcripts, irrespective of expression levels, are significantly more tissue specific than non-chimeric transcripts. The chimeric transcript we report here, showed a tissue expression pattern in the kidney. Also the parent transcripts showed tissue specific expression in the kidney which was also confirmed by the NRISH data. 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