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Analysis of cag-8: A novel poly(Q)-encoding gene in the mouse brain
BBRC Biochemical and Biophysical Research Communications 346 (2006) 1254–1260 www.elsevier.com/locate/ybbrc
Analysis of cag-8: A novel poly(Q)-encoding gene in the mouse brain q Jin Woo Ji, Hye Lim Yang, Sun Jung Kim
*
Department of Life Science, Dongguk University, Seoul 100-715, Republic of Korea Received 3 June 2006 Available online 14 June 2006
Abstract We identified 13 transcript isoforms of a trinucleotide-repeat-containing gene, cag-8, that is expressed almost exclusively in the mouse brain. The polypeptide deduced from the cDNA consists of 137 AAs, of which 74 are glutamines. The 130-kb gene is composed of 16 exons, from which at least 13 isoforms with variable UTRs are formed by alternative splicing. A strong positive cis-acting element was found in the neuroblastoma · glioma hybrid NG108-15 and human embryonic kidney HEK293 cell lines. The cag-8 protein was localized in the cytosol and in granular bodies within the nucleus. These findings indicate that cag-8 is a novel poly(Q)-encoding gene, the expression of which is confined primarily to the brain. 2006 Elsevier Inc. All rights reserved. Keywords: Mouse brain; Poly(Q); Trinucleotide repeat; Alternative splicing; 3 0 -Untranslated region
The large and continually growing number of genome sequencing projects provides an opportunity for us to greatly advance our understanding of genetics and disease. Although the entire sequence of the mouse genome is available and a comprehensive analysis of full-length cDNAs derived from the mouse genome has been completed [1,2], however, the function of many genes remains unknown. For example, of the approximately 30,000 genes in the mouse genome, the protein sequence is known for only approximately 20,000 and the gene ontology annotation has been completed for only 17,000 (mouse genome informatics database [http://www.informaticcs.jax.org]). Genes with short tandem-repeat segments are abundant in the mouse genome, as well as in all Swiss-Prot (14%), yeast (18%), and human proteins (28%) [3,4]. TNR sequences within the protein-coding region of the gene appear to undergo an abnormal expansion in individuals with neurological disorders [5–8]. Most TNR-associated diseases are caused
q Abbreviations: AA, amino acid; EST, expressed sequence tag; ORF, open reading frame; poly(Q), polyglutamine; RACE, rapid amplification of cDNA end; RT-PCR, reverse transcription-polymerase chain reaction; TNR, trinucleotide repeat; UTR, untranslated region. * Corresponding author. Fax: +82 2 2268 3975. E-mail address: [email protected] (S.J. Kim).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.029
by a CAG repeat [9]; of the 30 neurological disorders that are known to be caused by TNR expansion, 15 are induced by this structure. In mice and Drosophila, genes with CAG and CAA repeats encoding the AA glutamine are called opa repeats and are frequently found in homeobox genes or in other DNA-binding protein-encoding genes [10,11]. The CAG repeat sequence is dramatically enriched in the coding sequence, but is rarely found in intron sequences [12]. In mice, genes with a CAG-trinucleotide repeat sequence have been detected in brain tissue [13] and identified by searching databases of whole tissues [14]. In a previous study, we cloned a few CAG-repeat-containing genes from a mouse brain cDNA library [13]. Among them was the clone of cag-8, a novel expressed sequence tag (EST) containing 80 tandem CAG-repeats with a few base-pair (bp) interruptions, the function of which is yet to be elucidated. In this study, we cloned the full-length cDNA for this gene and found 13 new variants with alternative splicing patterns. We also analyzed its promoter, tissue specificity, and cellular localization. Materials and methods Sequence analysis. Splicing variants of cag-8 transcripts were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and
J.W. Ji et al. / Biochemical and Biophysical Research Communications 346 (2006) 1254–1260 subcloned into a pGEM-T vector (Promega, Madison, WI); the sequence of each transcript was then analyzed. The genomic sequence of cag-8 was identified using mouse BLAST (http://www.ncbi.nlm.nih.gov/genome/ seq/MmBlast.html). ESTs overlapping with U80891 were located with the general BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The open reading frame (ORF) and sequence alignments were identified using DNASIS.MAX version 2.0 for Windows (Miraibio, Alameda, CA). Cell culture. A mouse neuroblastoma · rat glioma hybrid cell line, NG108-15, was purchased from the American Type Culture Collection (ATCC; Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1· HAT (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (FBS). The human embryonic kidney HEK293 cell line was purchased form ATCC and grown in DMEM supplemented with 10% FBS. End-point RT-PCR analysis. The total RNA in ICR mice tissue was prepared using Trizol according to the supplier’s protocol (Gibco-BRL, Carlsbad, CA). Reverse transcription was carried out in 10 lg of total RNA using a reverse transcription kit (Promega). Tissue specificity and isoforms of the cag-8 transcript were determined by PCR using primers deduced from the EST AK045521. The primers—5 0 -GAGTCTCACT GTGCTGTCAT and 5 0 -ATACATCCCCTAAGTGCAGT—amplified a 562-bp fragment of AK045521. Glucose 3-phosphate dehydrogenase (G3PDH) primers were used to normalize cag-8 expression; their sequences are 5 0 -ACCACAGTCCATGCCATCAC and 5 0 -TCCACCAC CCTGTTGCTGTA, and they amplified a 500-bp cDNA fragment. A total of 25 cycles of the PCR was performed, each cycle being carried out at 94 C for 45 s, 62 C for 1 min, and then 72 C for 40 s. Quantitative real-time RT-PCR analysis. Reverse transcription was performed as described above for the end-point RT-PCR. PCRs were carried out using 100 ng of the template (cDNA), 300 nM of each forward and reverse primers, a dye-labeled TaqMan probe, and 1· PCR Master Mix (Applied Biosystems, Foster City, CA) in a volume of 20 lL. Each PCR plate contained triplicates of the test cDNA template and samples, which were used to construct a standard curve. Samples were amplified for 40 cycles in an ABI Prism 7300 Sequence Detection System (Applied Biosystems) with an initial melt at 95 C for 10 min, followed by 40 cycles, each carried out at 95 C for 15 s then 60 C for 1 min. The partial cycle giving a statistically significant increase in the cag-8 product was determined and normalized to G3PDH. The primer sequences used for cag-8 were as follows: forward primer, 5 0 -GCTTACACCTTTGGCATT TACCA; reverse primer, 5 0 -CAGGCACTATGAACTCTTGAAGCT; and probe, 5 0 -TCCAGCATAAAACCAAATAT. G3PDH was amplified using a probe and primers synthesized by Applied Biosystems. RACE analysis. To identify the 5 0 - and 3 0 -ends of the cag-8 transcript, we performed a rapid amplification of the cDNA end (RACE) using 2 mouse brain RACE kits (Roche, Basel, Switzerland; and Ambion, Austin, TX) according to the suppliers’ protocols. Cag-8-specific primers were used for the RACE analysis. For the 5 0 -RACE analysis, the primary PCR primer was 5 0 -CTGAAGGCTTCCGGAGCAAGCCTCC; the secondary PCR primer was 5 0 -GCAAGAGCCTCGTACAAGCTGAGAG. For the 3 0 -RACE analysis, the primary PCR primer was 5 0 -CCATGCCAATTA CAGTCAACCCAAG; the secondary PCR primer was 5 0 -ATCG ACCTAGGTGGGTAGGTCATTG. Luciferase assay. The upstream regions of the transcription start sites on cag-8 were amplified by nested PCR from the mouse chromosomal DNA and cloned into the pGL3Basic luciferase vector (Promega). We amplified the P1 and P3 regions, which are the upstream regions of exons 1 and 3, respectively. The primary PCR primers used to amplify P1 were 5 0 -CAGGAATGTGAAGAATCAGC and 5 0 -TGTACAGATGTGGGTC ACTC; the secondary PCR primers were 5 0 -TTTACCTGGCCATGGTG ACG and 5 0 -TCCTGAAGAGGAAGCTTCAG. The primary PCR primers used to amplify P3 were 5 0 -CCAACGTAATGACCAGAAGC and 5 0 -GAGGTAGCTTCAGATCATGC; the secondary PCR primers were 5 0 -TGCACAACATACAGGAGCAG and 5 0 -CAGAGTGGTTG AATAGGTAC. A total of 35 cycles of the PCR was performed, with each cycle conducted at 94 C for 30 s, 55 C for 1 min, and then 72 C for 2 min. The primer sequences for the nested PCR had XhoI and HindIII sites at each end to use in cloning into the pGL3Basic vector. A 2-lg
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volume of the recombinant luciferase expression vector was transiently transfected into 5 · 105 NG108-15 and HEK293 cells cultured on 100-mm culture dishes and harvested 36 h after transfection. Luciferase activity was measured in three independent cultures using a luciferase kit (Roche). A 1-lg volume of pCMV-bGal was cotransfected to normalize luciferase expression. Subcellular localization assay. Subcellular localization of the cag-8 protein was achieved by constructing a green fluorescent protein (GFP)/ cag-8 hybrid expressing vector and transiently transfecting it into NG10815 or HEK293 cells plated onto six-well glass-bottom tissue culture dishes. Expression of this protein was monitored under a confocal laser scanning fluorescence microscope. The fusion vector was prepared by ligating the PCR-derived product of cag-8 cDNA into the SacI and ApaI sites of the pGFP-C1 (Clontech, Mountain View, CA). The PCR primer sequences used to amplify cag-8 were 5 0 -GCTCCGGAAGCCTTCAGATG and 5 0 -GCACAGTGAGACTCTACATG.
Results Identification of alternatively spliced isoforms of cag-8 Cag-8 was identified using a mouse brain cDNA library in our previous study as an EST that contains 80 tandem CTGs with a few base replacements [13]. During our search for nucleotide databases to use in determining the fulllength cDNA for cag-8 by means of digital cloning, we found a 2311-bp EST, AK045521, overlapping with cag-8. Mouse genome BLAST revealed that the 2 ESTs originated from a single site on the mouse chromosome (GenBank Accession No. NT_039428). To determine the tissue specificity of cag-8, we carried out an RT-PCR procedure for the total RNA isolated from
Fig. 1. Endpoint RT-PCR of mouse tissues. (A) An RT-PCR was performed to identify the RNA of various mouse tissues using primer sets spanning exons 14–16: lane 1, heart; lane 2, embryo; lane 3, brain; lane 4, kidney; lane 5, liver; lane 6, lung; lane 7, testis; lane 8, muscle; lane 9, spleen. M is a molecular weight marker. The figure at the bottom is the result using G3PDH as a control. (B) A long-run agarose gel electrophoresis of RT-PCR products found in the brain. Lanes 1–4 represent the findings in the brain of 5-day, 7-day, 4-month, and 6-month-old mouse, respectively.
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canonical poly(A) signal sequence, AATAAA, appeared in exon 16, which was remarkably long (1566 bps). We synthesized the full length of the cDNA sequence by assembling portions of the EST and RACE sequences. The resulting cag-8 cDNAs were 2377–3528 bps long, depending on the length of the 3 0 -UTR of each isoform, and each harbored the same a 411-bp ORF (Fig. 3). Alignment with the genomic DNA sequence indicated that the gene has 16 exons and that 13 transcript isoforms were produced through variable combinations of these exons. The 5 0 UTR sequence was 192 bps long, and the ORF encoded a 137-AA polypeptide that included 74 glutamine residues, most of which were encoded by the CAG tandem repeat. The length of the 3 0 -UTR was variable, because the alternative splicing events were restricted to this region. Brain-specific expression of cag-8
Fig. 2. Multiple splicing variants of the mouse cag-8 gene. (A) Genomic organization of cag-8. The relative positions of exons (vertical lines) and introns (horizontal thin line) are shown. The boxes at the bottom indicate the inferred organization of the cag-8 exons. The exons are not drawn to scale. (B) Schematic diagrams of cag-8 transcripts identified by RT-PCR in brain and testis tissues are shown with their specific names. The exons and introns are represented by thick and thin bars, respectively. AK045521 is an EST that was found by means of a BLAST search and shows regions that overlap with a cag-8 transcript. Transcripts of r1–r5 were identified by 5 0 - and 3 0 -RACE analysis.
various mouse tissues. Cag-8 was expressed exclusively in the brain and testes (Fig. 1A); it was not found in embryonic tissue. Remarkably, the sizes of the transcripts were different in the brain compared with the testes. Further and more extensive RT-PCR analysis of brain and testis RNA revealed variable sizes of PCR products (Fig. 1B). When these products were sequence-analyzed after being subcloned into the pGEM-T vector, it was revealed that they were isoforms of the cag-8 transcripts created by alternative splicing (Fig. 2B). Thirteen isoforms were identified by comparing cDNA and genomic sequences. The structure of every transcript reflected the canonical GT/AG exon/intron junctions. The cag-8 gene encompassed approximately 130 kbs on chromosome 7 and contained 16 exons. Full-length cDNA analysis of cag-8 To obtain the full-length sequence of cag-8 cDNA, we explored the 5 0 - and 3 0 -RACE analysis findings further using RACE-ready brain cDNA libraries. The 5 0 -RACE analysis revealed two groups of transcripts with the 5 0 -end mapped near exons 1 and 3 (Fig. 2A). The 3 0 -RACE analysis revealed a single end with a poly(A) stretch. The
To examine the expression and tissue specificity of cag-8, quantitative real-time RT-PCR was carried out using the total RNA obtained from various mouse tissues. Primer sequences were designed to be complementary to regions spanning exons 14–16, which are common to almost all of the alternative forms of the transcripts. We found that cag-8 was expressed almost exclusively in the brain, with only minor expression in testis (Fig. 4A). The transcript was not detected in the embryonic tissues. To monitor in detail the expression of this gene in brain tissue, we performed real-time RT-PCR using total RNAs from various tissues in the mouse brain. Its expression was highest in the hypothalamus and relatively low in the midbrain and cerebellum. Moderate levels of expression were detected in other regions of the brain, including the cerebrum, hindbrain, and cerebral cortex. Promoter analysis and subcellular localization of cag-8 To verify that the upstream region of the end identified by RACE as being capable of functioning as a promoter region, we first tested regions approximately 2 kbs upstream of the two transcription start sites that were identified by RACE after it was linked to the pGL3 luciferase plasmid and transfected into the NG108-15 and HEK293 cells. We found that promoter activity was only induced by the sequence that was upstream of exon 1 (Fig. 5); the region close to exon 3 did not exhibit this function (data not shown). Various constructs containing the serially deleted DNA fragments upstream of exon 1 were examined, and a strong positive-acting element was identified in the region from 184 to 24 (Fig. 5). It has been reported that poly(Q)-containing proteins are localized primarily in cytosol (e.g., normal huntingtin [15] and ataxin-3 [16]) or in the nucleus (e.g., GRP-1 [17] and CBP [18]). We investigated NG108-15 and HEK293 cells that were transiently expressing cag-8/GFP to determine the localization of this protein. In both cell types, the recombinant protein was localized primarily in the
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Fig. 3. cDNA and deduced AA sequences of cag-8. The sequence of the cag-8 cDNA was deduced from the longest ORF, which was prepared by combining sequences identified through the EST and RACE analyses. The ORF of each transcription variant contains 192 bps of the 5 0 -UTR and 411 bps of the coding region in common and variable lengths for each 3 0 -UTR. The 3 0 -UTR sequence of a variant, v7 (see Fig. 2), which has the longest 3 0 -UTR is indicated. The CAG-repeat sequence codes for 41 tandem Q residues. The initiation and termination codons are indicated in bold. A poly(A) signal sequence (AATAAA) and the poly(A) are indicated bold and underlined. The sequence of cag-8 v7 was registered at the GenBank Database under the Accession No. DQ656357.
cytosol (Fig. 6); only a small amount was also found in the spot-like bodies in the nucleus. Discussion We have described the molecular cloning of a novel gene that is characterized primarily by an abundance of CAG repeats. The encoded protein, CAG-8, is remarkable because it contains mostly glutamine (54%) and undergoes extensive alternative splicing at its 3 0 -UTR. This gene seems to be unique in the mouse, because human or other mammalian homologs were not found by extensive BLAST searches. Many other poly(Q)-containing genes have been reported in mammals, but their precise function has not been determined yet. For example, huntingtin alleles on normal chromosomes contain fewer than 30–34 CAG repeats, whereas chromosomes in individuals manifesting Huntington’s chorea contain more than 35–40 repeats [19,20]. In other cases, including GRP-1 which acts as a transcriptional factor in macrophages, poly(Q) motifs are interspersed within histidine-rich regions [17]. An in silico approach was carried out to find poly(Q)-containing pep-
tides throughout the genome [21]. In cag-8, the poly(Q) stretch is interrupted by a few glutamic acids, cysteines, or leucines. It was known that the majority of repeat-containing proteins are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling [21]. Poly(Q)-containing proteins are especially involved in DNA and RNA binding, signaling, structural motif, and transport. The function of cag-8 remains obscure, however, as does the role of the poly(Q) segment. The cag-8 gene undergoes extensive splicing events in its 3 0 -UTR while it is being transcribed. Many mammalian genes also encode multiple splice variants at the 5 0 as well as the 3 0 -UTR. Alternative splicing at the 5 0 -UTR can play a role in determining tissue-specific expression of protein isoforms (e.g., in the glucocorticoid receptor) [22], or it can regulate translational efficiency through its effect on ribosome entry [23]. The 3 0 -UTRs of eukaryotic mRNAs are known to play a crucial role in the post-transcriptional regulation of gene expression by modulating nucleocytoplasmic mRNA transport, polyadenylation status, subcellular targeting, translation
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Fig. 4. Tissue-specific expression of the mouse cag-8 gene. To determine the tissue specificity and relative levels of expression for cag-8, real-time PCR was performed for the RNA of various mouse tissues. The expression level is indicated as the average of three independent reactions, with a standard error bar, after the data had been normalized with G3PDH. (A) The results for the following tissues: lane 1, brain; land 2, embryo; lane 3, heart; lane 4, kidney; lane 5, liver; lane 6, lung; lane 7, skeletal muscle; lane 8, pancreas; lane 9, spleen; lane 10, testis. (B) The results for the following tissues: lane 1, cerebrum; lane 2, cerebellum; lane 3, midbrain; lane 4, hindbrain; lane 5, frontal cortex; lane 6, posterior cortex; lane 7, hypothalamus; lane 8, hippocampus; lane 9, olfactory bulb; lane 10, thalamus.
Fig. 5. Identification of promoter elements responsible for the expression of cag-8 in transfected cells. Different parts of the upstream region of cag-8 (from 2035 to +139) were amplified by PCR and inserted into the pGL3Basic reporter plasmid, and relative luciferase activities were determined with cell extracts from transiently transfected cell lines. The arrow denotes the putative transcriptional initiation site. Each experiment was performed at least three times and normalized with b-galactosidase activity.
efficiency, stability, and rate of degradation [24–26]. However, few cases have been reported that undergo extensive splicing at the 3 0 -UTR [24]. In total, 13 isoforms of transcripts with different UTRs were found in this study, but it is possible that more isoforms may be further found. Although the significance of the alternative mRNA splicing remains unclear, it is important to characterize the full spectrum of structurally distinct cag-8 transcripts in the brain and testis, where they are predominantly expressed. We have identified a specific region (184 to 24) that is located just upstream of exon 1 and is crucial for the promoter activity of cag-8 in the NG108-15 and HEK293 cells. In the NG108-15 cells, promoter activity involved a more complex pattern, not only within the positive-acting element (184 to 24), but also within a strongly negative-acting element (1004 to 486). This result suggests that the negative element is dominant over the positive one and causes cag-8 expression to decline to a near basal level. It is known that the neuron-specific expression of many neuronal genes is generally regulated by a silencer element [27,28]. Further study of the relationship between the positive and negative elements, as well as the tissue specificity of this gene, should provide useful information about the properties of these elements. The cag-8 protein was primarily found evenly distributed throughout the cytosol, but also appeared in the nucleus in the form of granular bodies. The subcellular localization of an artificially truncated form of this protein containing only Q was reported previously as being excluded from cell nuclei [29]. Poly(Q)-peptides containing a nuclear localization signal, however, allowed them to be localized to the nucleus of the cell. In this respect, it is notable that the cag-8/GFP fusion protein was localized both in the cytosol and in nuclear bodies, which contain pre-mRNA splicing factors and polyadenylated RNA [30]. Consistent with the nuclear localization of cag-8, it does not contain any plausible signal sequence, but does contain a putative nuclear localization signal, RNTRRRRRR, at its C terminus (AAs 122–130). An RT-PCR analysis revealed that cag-8 is expressed predominantly in brain tissue after birth and in the testis, although to a lesser degree. It was not detected in the embryo. In this respect, cag-8 is expressed in a very brain-specific manner. It is also considered to be in the category of rarely expressed genes, because its expression was not detected by in situ hybridization in brain sections or by Northern blot analysis; it was only detected by RT-PCR. We conclude that cag-8 encodes a poly(Q)-containing protein that is expressed almost exclusively and in very small amounts in mouse brain tissue and undergoes extensive alternative splicing in its 3 0 -UTR. Further research should be focused on elucidating the role of the multiple splicing variants and address the function of the protein, both in the nucleus and cytosol.
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Fig. 6. Localization of GFP/cag-8 fusion protein to the cytosol and to granular bodies in the nucleus. NG108-15 and HEK293 cells expressing GFP/cag-8 were washed with phosphate-buffered solution and examined under a confocal microscope (magnification, 200·). GFP/cag-8 was identified in the cytosol as well as in speckle-like granular bodies in the nucleus.
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Analysis of cag-8: A novel poly(Q)-encoding gene in the mouse brain q Jin Woo Ji, Hye Lim Yang, Sun Jung Kim
*
Department of Life Science, Dongguk University, Seoul 100-715, Republic of Korea Received 3 June 2006 Available online 14 June 2006
Abstract We identified 13 transcript isoforms of a trinucleotide-repeat-containing gene, cag-8, that is expressed almost exclusively in the mouse brain. The polypeptide deduced from the cDNA consists of 137 AAs, of which 74 are glutamines. The 130-kb gene is composed of 16 exons, from which at least 13 isoforms with variable UTRs are formed by alternative splicing. A strong positive cis-acting element was found in the neuroblastoma · glioma hybrid NG108-15 and human embryonic kidney HEK293 cell lines. The cag-8 protein was localized in the cytosol and in granular bodies within the nucleus. These findings indicate that cag-8 is a novel poly(Q)-encoding gene, the expression of which is confined primarily to the brain. 2006 Elsevier Inc. All rights reserved. Keywords: Mouse brain; Poly(Q); Trinucleotide repeat; Alternative splicing; 3 0 -Untranslated region
The large and continually growing number of genome sequencing projects provides an opportunity for us to greatly advance our understanding of genetics and disease. Although the entire sequence of the mouse genome is available and a comprehensive analysis of full-length cDNAs derived from the mouse genome has been completed [1,2], however, the function of many genes remains unknown. For example, of the approximately 30,000 genes in the mouse genome, the protein sequence is known for only approximately 20,000 and the gene ontology annotation has been completed for only 17,000 (mouse genome informatics database [http://www.informaticcs.jax.org]). Genes with short tandem-repeat segments are abundant in the mouse genome, as well as in all Swiss-Prot (14%), yeast (18%), and human proteins (28%) [3,4]. TNR sequences within the protein-coding region of the gene appear to undergo an abnormal expansion in individuals with neurological disorders [5–8]. Most TNR-associated diseases are caused
q Abbreviations: AA, amino acid; EST, expressed sequence tag; ORF, open reading frame; poly(Q), polyglutamine; RACE, rapid amplification of cDNA end; RT-PCR, reverse transcription-polymerase chain reaction; TNR, trinucleotide repeat; UTR, untranslated region. * Corresponding author. Fax: +82 2 2268 3975. E-mail address: [email protected] (S.J. Kim).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.029
by a CAG repeat [9]; of the 30 neurological disorders that are known to be caused by TNR expansion, 15 are induced by this structure. In mice and Drosophila, genes with CAG and CAA repeats encoding the AA glutamine are called opa repeats and are frequently found in homeobox genes or in other DNA-binding protein-encoding genes [10,11]. The CAG repeat sequence is dramatically enriched in the coding sequence, but is rarely found in intron sequences [12]. In mice, genes with a CAG-trinucleotide repeat sequence have been detected in brain tissue [13] and identified by searching databases of whole tissues [14]. In a previous study, we cloned a few CAG-repeat-containing genes from a mouse brain cDNA library [13]. Among them was the clone of cag-8, a novel expressed sequence tag (EST) containing 80 tandem CAG-repeats with a few base-pair (bp) interruptions, the function of which is yet to be elucidated. In this study, we cloned the full-length cDNA for this gene and found 13 new variants with alternative splicing patterns. We also analyzed its promoter, tissue specificity, and cellular localization. Materials and methods Sequence analysis. Splicing variants of cag-8 transcripts were amplified by reverse transcription-polymerase chain reaction (RT-PCR) and
J.W. Ji et al. / Biochemical and Biophysical Research Communications 346 (2006) 1254–1260 subcloned into a pGEM-T vector (Promega, Madison, WI); the sequence of each transcript was then analyzed. The genomic sequence of cag-8 was identified using mouse BLAST (http://www.ncbi.nlm.nih.gov/genome/ seq/MmBlast.html). ESTs overlapping with U80891 were located with the general BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). The open reading frame (ORF) and sequence alignments were identified using DNASIS.MAX version 2.0 for Windows (Miraibio, Alameda, CA). Cell culture. A mouse neuroblastoma · rat glioma hybrid cell line, NG108-15, was purchased from the American Type Culture Collection (ATCC; Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1· HAT (Invitrogen, Carlsbad, CA) and 10% fetal bovine serum (FBS). The human embryonic kidney HEK293 cell line was purchased form ATCC and grown in DMEM supplemented with 10% FBS. End-point RT-PCR analysis. The total RNA in ICR mice tissue was prepared using Trizol according to the supplier’s protocol (Gibco-BRL, Carlsbad, CA). Reverse transcription was carried out in 10 lg of total RNA using a reverse transcription kit (Promega). Tissue specificity and isoforms of the cag-8 transcript were determined by PCR using primers deduced from the EST AK045521. The primers—5 0 -GAGTCTCACT GTGCTGTCAT and 5 0 -ATACATCCCCTAAGTGCAGT—amplified a 562-bp fragment of AK045521. Glucose 3-phosphate dehydrogenase (G3PDH) primers were used to normalize cag-8 expression; their sequences are 5 0 -ACCACAGTCCATGCCATCAC and 5 0 -TCCACCAC CCTGTTGCTGTA, and they amplified a 500-bp cDNA fragment. A total of 25 cycles of the PCR was performed, each cycle being carried out at 94 C for 45 s, 62 C for 1 min, and then 72 C for 40 s. Quantitative real-time RT-PCR analysis. Reverse transcription was performed as described above for the end-point RT-PCR. PCRs were carried out using 100 ng of the template (cDNA), 300 nM of each forward and reverse primers, a dye-labeled TaqMan probe, and 1· PCR Master Mix (Applied Biosystems, Foster City, CA) in a volume of 20 lL. Each PCR plate contained triplicates of the test cDNA template and samples, which were used to construct a standard curve. Samples were amplified for 40 cycles in an ABI Prism 7300 Sequence Detection System (Applied Biosystems) with an initial melt at 95 C for 10 min, followed by 40 cycles, each carried out at 95 C for 15 s then 60 C for 1 min. The partial cycle giving a statistically significant increase in the cag-8 product was determined and normalized to G3PDH. The primer sequences used for cag-8 were as follows: forward primer, 5 0 -GCTTACACCTTTGGCATT TACCA; reverse primer, 5 0 -CAGGCACTATGAACTCTTGAAGCT; and probe, 5 0 -TCCAGCATAAAACCAAATAT. G3PDH was amplified using a probe and primers synthesized by Applied Biosystems. RACE analysis. To identify the 5 0 - and 3 0 -ends of the cag-8 transcript, we performed a rapid amplification of the cDNA end (RACE) using 2 mouse brain RACE kits (Roche, Basel, Switzerland; and Ambion, Austin, TX) according to the suppliers’ protocols. Cag-8-specific primers were used for the RACE analysis. For the 5 0 -RACE analysis, the primary PCR primer was 5 0 -CTGAAGGCTTCCGGAGCAAGCCTCC; the secondary PCR primer was 5 0 -GCAAGAGCCTCGTACAAGCTGAGAG. For the 3 0 -RACE analysis, the primary PCR primer was 5 0 -CCATGCCAATTA CAGTCAACCCAAG; the secondary PCR primer was 5 0 -ATCG ACCTAGGTGGGTAGGTCATTG. Luciferase assay. The upstream regions of the transcription start sites on cag-8 were amplified by nested PCR from the mouse chromosomal DNA and cloned into the pGL3Basic luciferase vector (Promega). We amplified the P1 and P3 regions, which are the upstream regions of exons 1 and 3, respectively. The primary PCR primers used to amplify P1 were 5 0 -CAGGAATGTGAAGAATCAGC and 5 0 -TGTACAGATGTGGGTC ACTC; the secondary PCR primers were 5 0 -TTTACCTGGCCATGGTG ACG and 5 0 -TCCTGAAGAGGAAGCTTCAG. The primary PCR primers used to amplify P3 were 5 0 -CCAACGTAATGACCAGAAGC and 5 0 -GAGGTAGCTTCAGATCATGC; the secondary PCR primers were 5 0 -TGCACAACATACAGGAGCAG and 5 0 -CAGAGTGGTTG AATAGGTAC. A total of 35 cycles of the PCR was performed, with each cycle conducted at 94 C for 30 s, 55 C for 1 min, and then 72 C for 2 min. The primer sequences for the nested PCR had XhoI and HindIII sites at each end to use in cloning into the pGL3Basic vector. A 2-lg
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volume of the recombinant luciferase expression vector was transiently transfected into 5 · 105 NG108-15 and HEK293 cells cultured on 100-mm culture dishes and harvested 36 h after transfection. Luciferase activity was measured in three independent cultures using a luciferase kit (Roche). A 1-lg volume of pCMV-bGal was cotransfected to normalize luciferase expression. Subcellular localization assay. Subcellular localization of the cag-8 protein was achieved by constructing a green fluorescent protein (GFP)/ cag-8 hybrid expressing vector and transiently transfecting it into NG10815 or HEK293 cells plated onto six-well glass-bottom tissue culture dishes. Expression of this protein was monitored under a confocal laser scanning fluorescence microscope. The fusion vector was prepared by ligating the PCR-derived product of cag-8 cDNA into the SacI and ApaI sites of the pGFP-C1 (Clontech, Mountain View, CA). The PCR primer sequences used to amplify cag-8 were 5 0 -GCTCCGGAAGCCTTCAGATG and 5 0 -GCACAGTGAGACTCTACATG.
Results Identification of alternatively spliced isoforms of cag-8 Cag-8 was identified using a mouse brain cDNA library in our previous study as an EST that contains 80 tandem CTGs with a few base replacements [13]. During our search for nucleotide databases to use in determining the fulllength cDNA for cag-8 by means of digital cloning, we found a 2311-bp EST, AK045521, overlapping with cag-8. Mouse genome BLAST revealed that the 2 ESTs originated from a single site on the mouse chromosome (GenBank Accession No. NT_039428). To determine the tissue specificity of cag-8, we carried out an RT-PCR procedure for the total RNA isolated from
Fig. 1. Endpoint RT-PCR of mouse tissues. (A) An RT-PCR was performed to identify the RNA of various mouse tissues using primer sets spanning exons 14–16: lane 1, heart; lane 2, embryo; lane 3, brain; lane 4, kidney; lane 5, liver; lane 6, lung; lane 7, testis; lane 8, muscle; lane 9, spleen. M is a molecular weight marker. The figure at the bottom is the result using G3PDH as a control. (B) A long-run agarose gel electrophoresis of RT-PCR products found in the brain. Lanes 1–4 represent the findings in the brain of 5-day, 7-day, 4-month, and 6-month-old mouse, respectively.
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canonical poly(A) signal sequence, AATAAA, appeared in exon 16, which was remarkably long (1566 bps). We synthesized the full length of the cDNA sequence by assembling portions of the EST and RACE sequences. The resulting cag-8 cDNAs were 2377–3528 bps long, depending on the length of the 3 0 -UTR of each isoform, and each harbored the same a 411-bp ORF (Fig. 3). Alignment with the genomic DNA sequence indicated that the gene has 16 exons and that 13 transcript isoforms were produced through variable combinations of these exons. The 5 0 UTR sequence was 192 bps long, and the ORF encoded a 137-AA polypeptide that included 74 glutamine residues, most of which were encoded by the CAG tandem repeat. The length of the 3 0 -UTR was variable, because the alternative splicing events were restricted to this region. Brain-specific expression of cag-8
Fig. 2. Multiple splicing variants of the mouse cag-8 gene. (A) Genomic organization of cag-8. The relative positions of exons (vertical lines) and introns (horizontal thin line) are shown. The boxes at the bottom indicate the inferred organization of the cag-8 exons. The exons are not drawn to scale. (B) Schematic diagrams of cag-8 transcripts identified by RT-PCR in brain and testis tissues are shown with their specific names. The exons and introns are represented by thick and thin bars, respectively. AK045521 is an EST that was found by means of a BLAST search and shows regions that overlap with a cag-8 transcript. Transcripts of r1–r5 were identified by 5 0 - and 3 0 -RACE analysis.
various mouse tissues. Cag-8 was expressed exclusively in the brain and testes (Fig. 1A); it was not found in embryonic tissue. Remarkably, the sizes of the transcripts were different in the brain compared with the testes. Further and more extensive RT-PCR analysis of brain and testis RNA revealed variable sizes of PCR products (Fig. 1B). When these products were sequence-analyzed after being subcloned into the pGEM-T vector, it was revealed that they were isoforms of the cag-8 transcripts created by alternative splicing (Fig. 2B). Thirteen isoforms were identified by comparing cDNA and genomic sequences. The structure of every transcript reflected the canonical GT/AG exon/intron junctions. The cag-8 gene encompassed approximately 130 kbs on chromosome 7 and contained 16 exons. Full-length cDNA analysis of cag-8 To obtain the full-length sequence of cag-8 cDNA, we explored the 5 0 - and 3 0 -RACE analysis findings further using RACE-ready brain cDNA libraries. The 5 0 -RACE analysis revealed two groups of transcripts with the 5 0 -end mapped near exons 1 and 3 (Fig. 2A). The 3 0 -RACE analysis revealed a single end with a poly(A) stretch. The
To examine the expression and tissue specificity of cag-8, quantitative real-time RT-PCR was carried out using the total RNA obtained from various mouse tissues. Primer sequences were designed to be complementary to regions spanning exons 14–16, which are common to almost all of the alternative forms of the transcripts. We found that cag-8 was expressed almost exclusively in the brain, with only minor expression in testis (Fig. 4A). The transcript was not detected in the embryonic tissues. To monitor in detail the expression of this gene in brain tissue, we performed real-time RT-PCR using total RNAs from various tissues in the mouse brain. Its expression was highest in the hypothalamus and relatively low in the midbrain and cerebellum. Moderate levels of expression were detected in other regions of the brain, including the cerebrum, hindbrain, and cerebral cortex. Promoter analysis and subcellular localization of cag-8 To verify that the upstream region of the end identified by RACE as being capable of functioning as a promoter region, we first tested regions approximately 2 kbs upstream of the two transcription start sites that were identified by RACE after it was linked to the pGL3 luciferase plasmid and transfected into the NG108-15 and HEK293 cells. We found that promoter activity was only induced by the sequence that was upstream of exon 1 (Fig. 5); the region close to exon 3 did not exhibit this function (data not shown). Various constructs containing the serially deleted DNA fragments upstream of exon 1 were examined, and a strong positive-acting element was identified in the region from 184 to 24 (Fig. 5). It has been reported that poly(Q)-containing proteins are localized primarily in cytosol (e.g., normal huntingtin [15] and ataxin-3 [16]) or in the nucleus (e.g., GRP-1 [17] and CBP [18]). We investigated NG108-15 and HEK293 cells that were transiently expressing cag-8/GFP to determine the localization of this protein. In both cell types, the recombinant protein was localized primarily in the
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Fig. 3. cDNA and deduced AA sequences of cag-8. The sequence of the cag-8 cDNA was deduced from the longest ORF, which was prepared by combining sequences identified through the EST and RACE analyses. The ORF of each transcription variant contains 192 bps of the 5 0 -UTR and 411 bps of the coding region in common and variable lengths for each 3 0 -UTR. The 3 0 -UTR sequence of a variant, v7 (see Fig. 2), which has the longest 3 0 -UTR is indicated. The CAG-repeat sequence codes for 41 tandem Q residues. The initiation and termination codons are indicated in bold. A poly(A) signal sequence (AATAAA) and the poly(A) are indicated bold and underlined. The sequence of cag-8 v7 was registered at the GenBank Database under the Accession No. DQ656357.
cytosol (Fig. 6); only a small amount was also found in the spot-like bodies in the nucleus. Discussion We have described the molecular cloning of a novel gene that is characterized primarily by an abundance of CAG repeats. The encoded protein, CAG-8, is remarkable because it contains mostly glutamine (54%) and undergoes extensive alternative splicing at its 3 0 -UTR. This gene seems to be unique in the mouse, because human or other mammalian homologs were not found by extensive BLAST searches. Many other poly(Q)-containing genes have been reported in mammals, but their precise function has not been determined yet. For example, huntingtin alleles on normal chromosomes contain fewer than 30–34 CAG repeats, whereas chromosomes in individuals manifesting Huntington’s chorea contain more than 35–40 repeats [19,20]. In other cases, including GRP-1 which acts as a transcriptional factor in macrophages, poly(Q) motifs are interspersed within histidine-rich regions [17]. An in silico approach was carried out to find poly(Q)-containing pep-
tides throughout the genome [21]. In cag-8, the poly(Q) stretch is interrupted by a few glutamic acids, cysteines, or leucines. It was known that the majority of repeat-containing proteins are involved in processes that require the assembly of large, multiprotein complexes, such as transcription and signaling [21]. Poly(Q)-containing proteins are especially involved in DNA and RNA binding, signaling, structural motif, and transport. The function of cag-8 remains obscure, however, as does the role of the poly(Q) segment. The cag-8 gene undergoes extensive splicing events in its 3 0 -UTR while it is being transcribed. Many mammalian genes also encode multiple splice variants at the 5 0 as well as the 3 0 -UTR. Alternative splicing at the 5 0 -UTR can play a role in determining tissue-specific expression of protein isoforms (e.g., in the glucocorticoid receptor) [22], or it can regulate translational efficiency through its effect on ribosome entry [23]. The 3 0 -UTRs of eukaryotic mRNAs are known to play a crucial role in the post-transcriptional regulation of gene expression by modulating nucleocytoplasmic mRNA transport, polyadenylation status, subcellular targeting, translation
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Fig. 4. Tissue-specific expression of the mouse cag-8 gene. To determine the tissue specificity and relative levels of expression for cag-8, real-time PCR was performed for the RNA of various mouse tissues. The expression level is indicated as the average of three independent reactions, with a standard error bar, after the data had been normalized with G3PDH. (A) The results for the following tissues: lane 1, brain; land 2, embryo; lane 3, heart; lane 4, kidney; lane 5, liver; lane 6, lung; lane 7, skeletal muscle; lane 8, pancreas; lane 9, spleen; lane 10, testis. (B) The results for the following tissues: lane 1, cerebrum; lane 2, cerebellum; lane 3, midbrain; lane 4, hindbrain; lane 5, frontal cortex; lane 6, posterior cortex; lane 7, hypothalamus; lane 8, hippocampus; lane 9, olfactory bulb; lane 10, thalamus.
Fig. 5. Identification of promoter elements responsible for the expression of cag-8 in transfected cells. Different parts of the upstream region of cag-8 (from 2035 to +139) were amplified by PCR and inserted into the pGL3Basic reporter plasmid, and relative luciferase activities were determined with cell extracts from transiently transfected cell lines. The arrow denotes the putative transcriptional initiation site. Each experiment was performed at least three times and normalized with b-galactosidase activity.
efficiency, stability, and rate of degradation [24–26]. However, few cases have been reported that undergo extensive splicing at the 3 0 -UTR [24]. In total, 13 isoforms of transcripts with different UTRs were found in this study, but it is possible that more isoforms may be further found. Although the significance of the alternative mRNA splicing remains unclear, it is important to characterize the full spectrum of structurally distinct cag-8 transcripts in the brain and testis, where they are predominantly expressed. We have identified a specific region (184 to 24) that is located just upstream of exon 1 and is crucial for the promoter activity of cag-8 in the NG108-15 and HEK293 cells. In the NG108-15 cells, promoter activity involved a more complex pattern, not only within the positive-acting element (184 to 24), but also within a strongly negative-acting element (1004 to 486). This result suggests that the negative element is dominant over the positive one and causes cag-8 expression to decline to a near basal level. It is known that the neuron-specific expression of many neuronal genes is generally regulated by a silencer element [27,28]. Further study of the relationship between the positive and negative elements, as well as the tissue specificity of this gene, should provide useful information about the properties of these elements. The cag-8 protein was primarily found evenly distributed throughout the cytosol, but also appeared in the nucleus in the form of granular bodies. The subcellular localization of an artificially truncated form of this protein containing only Q was reported previously as being excluded from cell nuclei [29]. Poly(Q)-peptides containing a nuclear localization signal, however, allowed them to be localized to the nucleus of the cell. In this respect, it is notable that the cag-8/GFP fusion protein was localized both in the cytosol and in nuclear bodies, which contain pre-mRNA splicing factors and polyadenylated RNA [30]. Consistent with the nuclear localization of cag-8, it does not contain any plausible signal sequence, but does contain a putative nuclear localization signal, RNTRRRRRR, at its C terminus (AAs 122–130). An RT-PCR analysis revealed that cag-8 is expressed predominantly in brain tissue after birth and in the testis, although to a lesser degree. It was not detected in the embryo. In this respect, cag-8 is expressed in a very brain-specific manner. It is also considered to be in the category of rarely expressed genes, because its expression was not detected by in situ hybridization in brain sections or by Northern blot analysis; it was only detected by RT-PCR. We conclude that cag-8 encodes a poly(Q)-containing protein that is expressed almost exclusively and in very small amounts in mouse brain tissue and undergoes extensive alternative splicing in its 3 0 -UTR. Further research should be focused on elucidating the role of the multiple splicing variants and address the function of the protein, both in the nucleus and cytosol.
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Fig. 6. Localization of GFP/cag-8 fusion protein to the cytosol and to granular bodies in the nucleus. NG108-15 and HEK293 cells expressing GFP/cag-8 were washed with phosphate-buffered solution and examined under a confocal microscope (magnification, 200·). GFP/cag-8 was identified in the cytosol as well as in speckle-like granular bodies in the nucleus.
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