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Identification of new alternative splice events in the TCIRG1 gene in different human tissues
BBRC Biochemical and Biophysical Research Communications 330 (2005) 943–949 www.elsevier.com/locate/ybbrc
Identification of new alternative splice events in the TCIRG1 gene in different human tissues Anna S. Smirnova a, Andrey Morgun a,*, Natalia Shulzhenko a, Ismael D.C.G. Silva b, Maria Gerbase-DeLima a a
Immunogenetics Division, Pediatrics Department, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil b Gynecology Department, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil Received 7 March 2005 Available online 18 March 2005
Abstract Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice forms in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption. Ó 2005 Elsevier Inc. All rights reserved. Keywords: TCIRG1; Alternative splicing; Splice variants; Cytoplasmic RNA; RNA degradation
In eukaryotic cells, a single gene may give rise to various mRNA isoforms using mechanisms of alternative initiation, splicing, and termination. The mRNA isoforms could code for different products, increasing the protein diversity. Expression of splice variants may be specifically regulated in different tissues, and developmental and functional states [1]. Aberrant splicing may be associated with various disorders [2]. The human genome contains about 40,000 genes and, as estimated from expressed sequence tag (EST) analysis, at least 45% of them could be spliced alternatively [3]. However, the number of experimentally studied alternative forms is limited to about 1100 ones [4]. We applied EST analysis for a systematic identification of
*
Corresponding author. Fax: +55 11 5081 5028. E-mail address: [email protected] (A. Morgun).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.065
alternative splice events in the T cell immune regulator gene (TCIRG1). TCIRG1 is located on chromosome 11q13 and spans 12.5 kb [5]. Two transcript variants of this gene have been identified so far: transcript variant 1 (TV1), containing 20 exons, and transcript variant 2 (TV2), containing 15 exons [5]. TV2 transcription starts within exon 5 and continues through intron 5 of the gene; the last 14 exons are shared with TV1. Proteins encoded by TV1 and TV2 are named isoforms ‘‘a’’ and ‘‘b,’’ respectively. Isoform ‘‘a’’ represents the a3 subunit of the vacuolar proton pump, preferentially expressed in osteoclasts [6], and plays an important role in bone resorption. Mutations in TCIRG1 account for about 50% of the cases of infantile malignant osteopetrosis (IMO) [7–11]. Isoform ‘‘b’’ is a transmembrane molecule expressed on activated T cells [12]. It represents an inhibitory receptor that plays an essential role in the
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regulation of the immune response [13]. Our group demonstrated an association between acute rejection of cardiac transplants and increased intragraft expression of TCIRG1 in humans [14,15]. Recently, some new alternative splice events in TCIRG1 were described in patients with IMO [10,11,16]. Also, Susani et al. [11] identified retention of intron 5 in the TV1-like transcript in five normal human tissues. In the present study, we report the identification of various new alternative splice events in TCIRG1 in different tissues of normal individuals.
Materials and methods Bioinformatics. The EST database (dbEST) was searched against TV1 using the Basic Local Alignment Search Tool (BLAST) at NCBI [17] and the Human Genome Browser at UCSC [18]. Protein transmembrane domains were predicted using ExPASy [19] and TopPred [20]. RNA preparation. Twelve total RNA samples from 11 human tissues were purchased from Ambion (Austin, TX, USA) and Clontech (Palo Alto, CA, USA). For 32 samples from 17 tissues, total RNA was
isolated in our laboratory using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The tissue types are listed in Table 2. The RNA quality was assessed on an Agilent 2100 bioanalyzer using RNA 6000 Nano LabChip kit (both from Agilent Technologies, Waldbronn, Germany). Both total and cytoplasmic RNA were isolated from peripheral blood mononuclear cells (PBMC) obtained from 18 healthy volunteers. Cytoplasmic RNA was isolated using Cytoplasmic RNA reagent (Invitrogen). The study was approved by the Ethics Committee of the Federal University of Sa˜o Paulo. Reverse transcription-polymerase chain reaction. Complementary DNA (cDNA) was prepared in a final volume of 50 ll using 500 ng RNA, 400 U of SuperScript II Reverse Transcriptase, 10 ll of FirstStrand Buffer, 5 ll of 0.1 M DTT, 10 ll of 10 mM dNTP mixture (all from Invitrogen), 0.2 lg of oligo(dT) primer, and 50 U RNAguard RNase inhibitor (Amersham Biosciences, Uppsala, Sweden). This mixture was incubated at 42 °C for 60 min and then at 70 °C for 10 min. The efficiency of cDNA preparation was assessed by amplification of a 335 bp fragment of the RNA polymerase II subunit K (POLR2K) with primers 5 0 -GGAAACGCGGAGTGAGTTTT-3 0 and 5 0 -CTCCCGAAGATAAGGGGGAA-3 0 . Sequences of primers for amplification of TCIRG1 splice forms are available on request; primer locations are listed in Table 1. PCR was performed on 1 ll cDNA using Eppendorf MasterMix (Eppendorf, Hamburg, Germany). The conditions were 40 cycles of amplification at 94 °C for 45 s, at 63 °C for 30 s, and at 72 °C for 30–90 s (PTC-200 Thermocycler; MJ Research, Wattertown, MA, USA). To detect splice forms skipping exon 19, we
Table 1 Putative alternative splice events in TCIRG1 indicated by a search in dbEST, and locations of primers used for their verification by RT-PCR Alternative splicing event
GenBank Accession Nos.
Number of tissues in dbEST
Locations of primers (forward–reverse)
Use of a cryptic exon within intron 1 (from 674 to 844 bp of intron 1)
BM920218, BM148472, BM144582, BE242518, BE242310 BM920218, BI837894, BI910724, BI907308, BM805434, BF847722 AL555376 BG029109, BF893844, BI759558, AL552184, BQ083991, BQ083933 BG434897 AA477666, AA291912 BF873942 BM981456, AI457772, AI989635, BF224014 BF813056
2
Intron 1–exon 4
4
Exon 1–intron 5
1 5
Exon 4–exon 7 Intron 12–exon 14
1 1 1 4
Exon 12–intron 13 Intron 15–exon 18 Exon 15–intron 16 Exon 17–intron 18
1
Exon 17–intron 19
Retention of intron 5 Deletion of exon 6a Retention of intron 12 Partial retention of intron 13 (from 858 to 902 bp) Retention of intron 15 Retention of intron 16a Retention of intron 18 Retention of intron 19a a
Splice event not confirmed in our study.
Table 2 Detection of nine TCIRG1 splice forms in different human tissues by RT-PCRa Number of splice forms detected
Tissues
9
Brain (2), colon, fallopian tube, liver, lung, breast (2), breast cancer (1)b, peripheral blood mononuclear cells (PBMC) (2)b, placenta (2)b, prostatec, skeletal muscle, testicle, thyroidc, tonsil, tracheac Adrenal gland, gallbladder, kidney, breast cancer (1)b, ovary, PBMC (1)b, placenta (3)b, uterine cervix (2)b, uterine cervical cancer (1)b, vaginal tunic of testes Adipose tissue, duodenum, peripheral nerve, spleen (4)b, stomach, uterine cervix (1)b, uterine cervical cancer (2)b, vaginal mucosa
8 <8
a Numbers in parenthesis indicate the number of samples from the same tissue that showed this splicing profile; when there is no number, only one sample was studied. b Different samples of the same tissue demonstrated different splicing profiles. c Pooled samples.
A.S. Smirnova et al. / Biochemical and Biophysical Research Communications 330 (2005) 943–949 used competitive PCR with selective enzymatic depletion of forms including exon 19 [21]: after 20 PCR cycles, samples were treated with restriction enzyme NlaIII and then amplified 27 cycles more (Fig. 1B). To detect the splice form with retention of intron 18, concomitant amplification of TCIRG1 DNA was precluded by treating with BstXI. Reverse transcription-polymerase chain reaction (RT-PCR) products were separated by electrophoresis in ethidium bromide-stained 2% agarose gels and images were captured under ultraviolet light with the Kodak Digital Science—EDAS 120 system (Eastman Kodak, Rochester, NY, USA). For semi-quantitative RT-PCR, band intensities were analyzed with the Kodak Digital Science 1D program (Eastman Kodak). The products to be sequenced were recovered from the gel using GFX PCR DNA and the Gel Band Purification kit (Amersham Biosciences). Sequencing was performed with a BigDye Terminator Cycle Sequencing kit (v3.1) on an ABI Prism 3100 Genetic Analyzer (both from Applied Biosystems, Foster city, CA, USA). GenBank accession numbers. Accession numbers for TCIRG1 transcript variants 1 and 2 are NM_006019 and NM_006053, respectively. We deposited the sequences of the new TCIRG1 variants in GenBank with the following accession numbers: transcript variant 3 (skipping of exon 19), AF497545; transcript variant 4 (a cryptic exon in intron 1), AY708388; transcript variant 5 (retention
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of intron 12), AY548968; transcript variant 6 (partial retention of intron 13), AY548969; transcript variant 7 (retention of intron 15), AY548970; transcript variant 8 (retention of intron 18), AY548971; transcript variant 9 (partial retention of intron 18 and skipping of exon 19), AY548972; transcript variant 10 (retention of intron 5), AY708389. Statistical analyses. The Spearman correlation coefficient was used to determine correlation between variables. Splice form expression levels between cytoplasmic and total RNA were compared using the paired t test.
Results Identification and validation of putative alternative splice events in TCIRG1 Searching in dbEST identified 200 ESTs with homology to TCIRG1. Twenty-three ESTs contained putative alternative splice events, comprising eight types of insertions and one deletion (Table 1). Most of the ESTs spanned from four to seven exons of
Fig. 1. Alternative splice events in TCIRG1 transcripts. The structure of the exons and introns of the gene is shown at the top. Boxes and lines represent exons and introns, respectively. The arrows mark primer positions. (A) Alternative splice events in the region between exon 1 and exon 7: (a) transcript variant 1, (b) transcript variant 2, and (c) splice form with retention of intron 5. (B) Alternative splice events in the region between exon 18 and exon 20: (a) transcript variants 1 and 2, (b) splice form with exclusion of exon 19, (c) splice form with full retention of intron 18, and (d) splice form with retention of intron 18 up to position 170+ and exclusion of exon 19. The restriction enzyme NlaIII was used for depletion of transcripts including exon 19 during competitive RT-PCR.
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TCIRG1 and contained only one alternative splice event. We searched for the nine putative splice events (Table 1) in 44 samples from 28 human tissues by RT-PCR. Six splice forms yielded products of expected sizes and were sequenced. Validated alternative splice events were the following: use of a cryptic exon within intron 1 (CrypEx_Int1), partial retention of intron 13 (Part_Ret_Int13), and full retention of introns 5, 12, 15 or 18 (Ret_Int5, Ret_Int12, Ret_Int15, Ret_Int18). Detection of the previously described TCIRG1 splice forms To detect transcripts using the TV1 transcription start site, we designed primers in exons 1 and 7 (TV1 ex1_7) (Fig. 1A (a)). TV2 uses an alternative transcription start site in exon 5 and includes intron 5 (Fig. 1A (b)); since intron 5 could be included in the longer splice form (Fig. 1A (c)), the separate identification of TV2 was not possible. In the present study, we also searched for the three alternative splice forms previously described in one patient with IMO [16]. A splice form with retention of intron 19 was verified within others indicated by the dbEST search, as described in Table 1. The form lacking exon 19 (Del_Ex19) (Fig 1B (b)) was detected by amplification with primers located in exons 18 and 20, and the form with extension of exon 18 to position +170 in authentic intron 18 and skipping of exon 19 (Part_Ret_Int18+Del_ex19) (Fig. 1B (d)) was detected by amplification with primers located in intron 18 and exon 20. The more highly expressed forms including exon 19 (Fig. 1B (a) and (c)) were depleted by digestion with NlaIII. Tissue distribution of the TCIRG1 splice forms Amplification of the region between exons 17 and 20, common for TV1 and TV2, in 44 samples from 28 human tissues (Table 2) demonstrated that the gene is ubiquitously expressed. We studied tissue distribution of the following nine TCIRG1 splice forms: TV1 ex1_7, CrypEx_Int1, Part_Ret_Int13, Ret_Int5, Ret_Int12, Ret_Int15, Ret_Int18, Del_Ex19, and Part_Ret_Int18 + Del_ex19. As shown in Table 2, 19 samples from 15 tissues demonstrated all of these nine splice forms, 13 samples demonstrated eight splice forms, and 12 less than eight splice forms. In some cases, samples from the same tissue showed different numbers of splice forms (Table 2). We later tested whether the number of detected splice forms is related to RNA quality. The mean 28S/18S rRNA ratio and the mean percentage of 28S and 18S rRNA area in our samples were 1.0 ± 0.6 and 15.6 ± 12.8, respectively. These two characteristics correlated well (r = 0.74, p < 0.0001). We defined the
Fig. 2. Relationship between RNA quality index (x) and number of TCIRG1 splice forms (y) detected in a sample. The RNA quality index is the sum of the 28S/18S rRNA ratio and the percentage of rRNA area of a sample, both expressed as percentages of the mean of our group.
RNA quality index as the sum of the 28S/18S rRNA ratio and the percentage of rRNA area of a sample, both expressed as percentages of the mean for the group (Fig. 2). We observed correlation between RNA quality index and the number of splice forms detected (r = 0.59, p < 0.001) (Fig. 2). Verification of the TCIRG1 splice forms in cytoplasmic RNA of PBMC Expression levels of the nine TCIRG1 splice forms were evaluated by semi-quantitative RT-PCR in both total and cytoplasmic RNA from 18 PBMC samples. Six splice forms, i.e., CrypEx_Int1, Ret_Int12, Ret_Int15, Del_Ex19, Part_Ret_Int18 + Del_ex19, and TV1 ex1_7, demonstrated similar (less than twofold difference) levels of expression in cytoplasmic and total RNA samples. The other three splice forms, Ret_Int5, Part_Ret_Int13, and Ret_Int18, were represented in cytoplasmic RNA at significantly lower levels (the median difference was more than 4, p < 0.001, as illustrated in Figs. 3A–C). Analysis of the open reading frame (ORF) The TV1 and TV2 translations initiate in exons 2 and 7 of the gene, respectively, and terminate in exon 20 [5].
Fig. 3. TCIRG1 splice forms with differential expression in cytoplasmic (C) and total (T) RNA: example of detection in one representative PBMC sample. (A) Splice form with retention of intron 5. (B) Splice form with partial retention of intron 13. (C) Splice form with retention of intron 18. (D) POLR2K as internal control.
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Fig. 4. Schematic presentation of the TCIRG1 isoform ‘‘a’’ topology. Arrows 1–5 indicate sites of protein truncation due to the appearance of the premature stop codon for retention of introns 12, 13, 15, 18 or exclusion of exon 19, respectively.
The three-dimensional topology of the corresponding protein isoforms has not yet been established experimentally. Different methods for the prediction of transmembrane helix topology based on hydrophobicity analysis of the protein sequence predict 7 (ExPASy) to 12 (TopPred) transmembrane a-helices for isoform ‘‘a.’’ Based on the experimental topology model of a yeast TCIRG1 homolog Vph1p [22], the isoform ‘‘a’’ could present a transmembrane region with nine a-helices (Fig. 4). The splice event CrypEx_Int1 did not change the ORF. Ret_Int5 in the full-length transcript results in a product similar to both TV1 and TV2, and does not allow prediction of its translation start site. Ret_Int12, Part_Ret_Int13, Ret_Int15 or Ret_Int18 generates a premature stop codon within the sequence corresponding to the intron, and Del_Ex19, within exon 20. Therefore, these five splice events would lead to the production of shortened proteins containing 3, 3, 5, 7, and 7 transmembrane domains, respectively (Fig. 4).
Discussion In the present study, we searched for alternative splice events in TCIRG1 in different human tissues. We validated six new alternative splice events indicated by a search in dbEST: CrypEx_Int1, Ret_Int5, Ret_Int12, Part_Ret_Int13, Ret_Int15, and Ret_Int18. They were observed in most of the 28 human tissues studied. Three unconfirmed putative splice events were indicated by single ESTs, and the failure in detecting them may be related to the low abundance or low frequency of these forms. We also investigated tissue distribution of the previously reported TCIRG1 splice forms. First, we verified the tissue distribution of TV1. The previous data about its expression were controversial: Li et al. [6], showed osteoclast-specific expression of the TV1 by Northern blot, and this finding was cited by several authors [7,10,16]; however, Scott and Chapman [23] showed ubiquitous distribution of TV1 by RT-PCR. Using RT-PCR, we also observed the product TV1 ex1_7 in
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most tissues. The difference between the results obtained by RT-PCR and Northern blot could be explained by the lower sensitivity of the latter. The data about almost exclusive expression of TV2 in immune tissues were also obtained by Northern blot [12], and, therefore, need further confirmation. Unfortunately, we were not able to design a strategy for specific amplification of this variant due to its high similarity to the other longer transcript (Fig. 1A). Next, we studied three TCIRG1 splice events, namely Ret_Int19, Del_Ex19, and Part_Ret_Int18 + Del_ex19, described as aberrant, related to IMO [16]. Although Ret_Int19 was not detected in any of our samples, the two other splice events were observed by us in various tissues of healthy individuals. Thus, we studied the nine alternative splice forms of the TCIRG1 gene and demonstrated that they occur in most tissues. However, some of the splice forms were not detected in a number of samples. The RNA quality in our samples varied strongly since it depends on tissue type [24,25] and RNA isolation method [26]. We observed that the number of splice events detected in our samples correlated with the RNA degradation level and therefore it was not possible to perform a precise analysis of their tissue-specific distribution. However, the expression analysis suggests that tissue distribution of the TCIRG1 splice forms is nearly ubiquitous. To verify whether TCIRG1 transcripts subjected to these alternative splice events could be potentially translated, we searched for the TCIRG1 splice forms in cytoplasmic RNA of PBMC samples. Six splice forms demonstrated similar levels of expression in cytoplasmic and total RNA, and therefore they are efficiently exported from the nucleus and could not be splicing intermediates. Three splice forms—with Ret_Int5, Part_Ret_Int13, and Ret_Int18—were hardly detected in cytoplasmic RNA. Although forms with simple intron retention could represent splicing intermediates, Part_Ret_Int13 uses an alternative acceptor splice site and, therefore, represents a bona fide alternative splice event. The first possible explanation for a low presentation of a splice form in cytoplasm is depletion by nonsense-mediated mRNA decay [27]. However, not all TCIRG1 splice forms generating premature stop codons are depleted. The second explanation could be regulation at the level of the nuclear export [28]. Recently, differential nucleocytoplasmic distribution of splice variants was also demonstrated for the human gene ADAM33 [29], but the relationship between alternative splicing pathways, RNA degradation, and nuclear export has not yet been studied. Also, it has been recently reported that nucleocytoplasmic distribution of mRNA could be regulated in a tissuespecific manner [30]. Therefore, the nucleocytoplasmic distribution of TCIRG1 splice forms warrants studies in other tissues.
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Our data indicate that splicing in TCIRG1 is extremely complex. Amplifications with primers specific for different alternative splice events yielded multiple bands, suggesting that alternative splice events in the gene may occur in various combinations (data not shown). The maximum number of possible combinations taking into account the nine splice types is 128. However, CrypEx_Int1 and Ret_Int5 did not modify the protein, and all the other alternative splice events identified in this study led to the appearance of the premature stop codon near the splice event. Thus, the amino acid sequence coded by a transcript containing multiple alternative splice events between exons 12 and 20 depends only on the first event. In summary, we validated six new alternative splice events in the TCIRG1 gene in various human tissues. We demonstrated that transcripts using the TV1 transcription start site and two splice forms previously described in one patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine TCIRG1 splice forms in cytoplasmic RNA of PBMC samples showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption.
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments We thank Dr. Emmanuel Dias Neto for helpful discussion of the manuscript and Dr. Paulo S.L. de Oliveira for help with the prediction of protein structure. We are grateful to Drs. Davimar M. Borducchi, Silvia Daher, and Mario Dolnikoff for help with sample collection. We thank Dr. Rui M.B. Maciel and Gustavo S. Guimara˜es for help with the DNA sequencing. This research was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Pesquisa (CNPq). A.S. Smirnova is the recipient of a FAPESP fellowship.
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Identification of new alternative splice events in the TCIRG1 gene in different human tissues Anna S. Smirnova a, Andrey Morgun a,*, Natalia Shulzhenko a, Ismael D.C.G. Silva b, Maria Gerbase-DeLima a a
Immunogenetics Division, Pediatrics Department, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil b Gynecology Department, Universidade Federal de Sa˜o Paulo (UNIFESP-EPM), Sa˜o Paulo, Brazil Received 7 March 2005 Available online 18 March 2005
Abstract Two transcript variants (TV) of the T cell immune regulator gene 1 (TCIRG1) have already been characterized. TV1 encodes a subunit of the osteoclast vacuolar proton pump and TV2 encodes a T cell inhibitory receptor. Based on the search in dbEST, we validated by RT-PCR six new alternative splice events in TCIRG1 in most of the 28 human tissues studied. In addition, we observed that transcripts using the TV1 transcription start site and two splice forms previously described in a patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine splice forms in cytoplasmic RNA of peripheral blood mononuclear cells showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption. Ó 2005 Elsevier Inc. All rights reserved. Keywords: TCIRG1; Alternative splicing; Splice variants; Cytoplasmic RNA; RNA degradation
In eukaryotic cells, a single gene may give rise to various mRNA isoforms using mechanisms of alternative initiation, splicing, and termination. The mRNA isoforms could code for different products, increasing the protein diversity. Expression of splice variants may be specifically regulated in different tissues, and developmental and functional states [1]. Aberrant splicing may be associated with various disorders [2]. The human genome contains about 40,000 genes and, as estimated from expressed sequence tag (EST) analysis, at least 45% of them could be spliced alternatively [3]. However, the number of experimentally studied alternative forms is limited to about 1100 ones [4]. We applied EST analysis for a systematic identification of
*
Corresponding author. Fax: +55 11 5081 5028. E-mail address: [email protected] (A. Morgun).
0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.03.065
alternative splice events in the T cell immune regulator gene (TCIRG1). TCIRG1 is located on chromosome 11q13 and spans 12.5 kb [5]. Two transcript variants of this gene have been identified so far: transcript variant 1 (TV1), containing 20 exons, and transcript variant 2 (TV2), containing 15 exons [5]. TV2 transcription starts within exon 5 and continues through intron 5 of the gene; the last 14 exons are shared with TV1. Proteins encoded by TV1 and TV2 are named isoforms ‘‘a’’ and ‘‘b,’’ respectively. Isoform ‘‘a’’ represents the a3 subunit of the vacuolar proton pump, preferentially expressed in osteoclasts [6], and plays an important role in bone resorption. Mutations in TCIRG1 account for about 50% of the cases of infantile malignant osteopetrosis (IMO) [7–11]. Isoform ‘‘b’’ is a transmembrane molecule expressed on activated T cells [12]. It represents an inhibitory receptor that plays an essential role in the
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regulation of the immune response [13]. Our group demonstrated an association between acute rejection of cardiac transplants and increased intragraft expression of TCIRG1 in humans [14,15]. Recently, some new alternative splice events in TCIRG1 were described in patients with IMO [10,11,16]. Also, Susani et al. [11] identified retention of intron 5 in the TV1-like transcript in five normal human tissues. In the present study, we report the identification of various new alternative splice events in TCIRG1 in different tissues of normal individuals.
Materials and methods Bioinformatics. The EST database (dbEST) was searched against TV1 using the Basic Local Alignment Search Tool (BLAST) at NCBI [17] and the Human Genome Browser at UCSC [18]. Protein transmembrane domains were predicted using ExPASy [19] and TopPred [20]. RNA preparation. Twelve total RNA samples from 11 human tissues were purchased from Ambion (Austin, TX, USA) and Clontech (Palo Alto, CA, USA). For 32 samples from 17 tissues, total RNA was
isolated in our laboratory using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The tissue types are listed in Table 2. The RNA quality was assessed on an Agilent 2100 bioanalyzer using RNA 6000 Nano LabChip kit (both from Agilent Technologies, Waldbronn, Germany). Both total and cytoplasmic RNA were isolated from peripheral blood mononuclear cells (PBMC) obtained from 18 healthy volunteers. Cytoplasmic RNA was isolated using Cytoplasmic RNA reagent (Invitrogen). The study was approved by the Ethics Committee of the Federal University of Sa˜o Paulo. Reverse transcription-polymerase chain reaction. Complementary DNA (cDNA) was prepared in a final volume of 50 ll using 500 ng RNA, 400 U of SuperScript II Reverse Transcriptase, 10 ll of FirstStrand Buffer, 5 ll of 0.1 M DTT, 10 ll of 10 mM dNTP mixture (all from Invitrogen), 0.2 lg of oligo(dT) primer, and 50 U RNAguard RNase inhibitor (Amersham Biosciences, Uppsala, Sweden). This mixture was incubated at 42 °C for 60 min and then at 70 °C for 10 min. The efficiency of cDNA preparation was assessed by amplification of a 335 bp fragment of the RNA polymerase II subunit K (POLR2K) with primers 5 0 -GGAAACGCGGAGTGAGTTTT-3 0 and 5 0 -CTCCCGAAGATAAGGGGGAA-3 0 . Sequences of primers for amplification of TCIRG1 splice forms are available on request; primer locations are listed in Table 1. PCR was performed on 1 ll cDNA using Eppendorf MasterMix (Eppendorf, Hamburg, Germany). The conditions were 40 cycles of amplification at 94 °C for 45 s, at 63 °C for 30 s, and at 72 °C for 30–90 s (PTC-200 Thermocycler; MJ Research, Wattertown, MA, USA). To detect splice forms skipping exon 19, we
Table 1 Putative alternative splice events in TCIRG1 indicated by a search in dbEST, and locations of primers used for their verification by RT-PCR Alternative splicing event
GenBank Accession Nos.
Number of tissues in dbEST
Locations of primers (forward–reverse)
Use of a cryptic exon within intron 1 (from 674 to 844 bp of intron 1)
BM920218, BM148472, BM144582, BE242518, BE242310 BM920218, BI837894, BI910724, BI907308, BM805434, BF847722 AL555376 BG029109, BF893844, BI759558, AL552184, BQ083991, BQ083933 BG434897 AA477666, AA291912 BF873942 BM981456, AI457772, AI989635, BF224014 BF813056
2
Intron 1–exon 4
4
Exon 1–intron 5
1 5
Exon 4–exon 7 Intron 12–exon 14
1 1 1 4
Exon 12–intron 13 Intron 15–exon 18 Exon 15–intron 16 Exon 17–intron 18
1
Exon 17–intron 19
Retention of intron 5 Deletion of exon 6a Retention of intron 12 Partial retention of intron 13 (from 858 to 902 bp) Retention of intron 15 Retention of intron 16a Retention of intron 18 Retention of intron 19a a
Splice event not confirmed in our study.
Table 2 Detection of nine TCIRG1 splice forms in different human tissues by RT-PCRa Number of splice forms detected
Tissues
9
Brain (2), colon, fallopian tube, liver, lung, breast (2), breast cancer (1)b, peripheral blood mononuclear cells (PBMC) (2)b, placenta (2)b, prostatec, skeletal muscle, testicle, thyroidc, tonsil, tracheac Adrenal gland, gallbladder, kidney, breast cancer (1)b, ovary, PBMC (1)b, placenta (3)b, uterine cervix (2)b, uterine cervical cancer (1)b, vaginal tunic of testes Adipose tissue, duodenum, peripheral nerve, spleen (4)b, stomach, uterine cervix (1)b, uterine cervical cancer (2)b, vaginal mucosa
8 <8
a Numbers in parenthesis indicate the number of samples from the same tissue that showed this splicing profile; when there is no number, only one sample was studied. b Different samples of the same tissue demonstrated different splicing profiles. c Pooled samples.
A.S. Smirnova et al. / Biochemical and Biophysical Research Communications 330 (2005) 943–949 used competitive PCR with selective enzymatic depletion of forms including exon 19 [21]: after 20 PCR cycles, samples were treated with restriction enzyme NlaIII and then amplified 27 cycles more (Fig. 1B). To detect the splice form with retention of intron 18, concomitant amplification of TCIRG1 DNA was precluded by treating with BstXI. Reverse transcription-polymerase chain reaction (RT-PCR) products were separated by electrophoresis in ethidium bromide-stained 2% agarose gels and images were captured under ultraviolet light with the Kodak Digital Science—EDAS 120 system (Eastman Kodak, Rochester, NY, USA). For semi-quantitative RT-PCR, band intensities were analyzed with the Kodak Digital Science 1D program (Eastman Kodak). The products to be sequenced were recovered from the gel using GFX PCR DNA and the Gel Band Purification kit (Amersham Biosciences). Sequencing was performed with a BigDye Terminator Cycle Sequencing kit (v3.1) on an ABI Prism 3100 Genetic Analyzer (both from Applied Biosystems, Foster city, CA, USA). GenBank accession numbers. Accession numbers for TCIRG1 transcript variants 1 and 2 are NM_006019 and NM_006053, respectively. We deposited the sequences of the new TCIRG1 variants in GenBank with the following accession numbers: transcript variant 3 (skipping of exon 19), AF497545; transcript variant 4 (a cryptic exon in intron 1), AY708388; transcript variant 5 (retention
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of intron 12), AY548968; transcript variant 6 (partial retention of intron 13), AY548969; transcript variant 7 (retention of intron 15), AY548970; transcript variant 8 (retention of intron 18), AY548971; transcript variant 9 (partial retention of intron 18 and skipping of exon 19), AY548972; transcript variant 10 (retention of intron 5), AY708389. Statistical analyses. The Spearman correlation coefficient was used to determine correlation between variables. Splice form expression levels between cytoplasmic and total RNA were compared using the paired t test.
Results Identification and validation of putative alternative splice events in TCIRG1 Searching in dbEST identified 200 ESTs with homology to TCIRG1. Twenty-three ESTs contained putative alternative splice events, comprising eight types of insertions and one deletion (Table 1). Most of the ESTs spanned from four to seven exons of
Fig. 1. Alternative splice events in TCIRG1 transcripts. The structure of the exons and introns of the gene is shown at the top. Boxes and lines represent exons and introns, respectively. The arrows mark primer positions. (A) Alternative splice events in the region between exon 1 and exon 7: (a) transcript variant 1, (b) transcript variant 2, and (c) splice form with retention of intron 5. (B) Alternative splice events in the region between exon 18 and exon 20: (a) transcript variants 1 and 2, (b) splice form with exclusion of exon 19, (c) splice form with full retention of intron 18, and (d) splice form with retention of intron 18 up to position 170+ and exclusion of exon 19. The restriction enzyme NlaIII was used for depletion of transcripts including exon 19 during competitive RT-PCR.
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TCIRG1 and contained only one alternative splice event. We searched for the nine putative splice events (Table 1) in 44 samples from 28 human tissues by RT-PCR. Six splice forms yielded products of expected sizes and were sequenced. Validated alternative splice events were the following: use of a cryptic exon within intron 1 (CrypEx_Int1), partial retention of intron 13 (Part_Ret_Int13), and full retention of introns 5, 12, 15 or 18 (Ret_Int5, Ret_Int12, Ret_Int15, Ret_Int18). Detection of the previously described TCIRG1 splice forms To detect transcripts using the TV1 transcription start site, we designed primers in exons 1 and 7 (TV1 ex1_7) (Fig. 1A (a)). TV2 uses an alternative transcription start site in exon 5 and includes intron 5 (Fig. 1A (b)); since intron 5 could be included in the longer splice form (Fig. 1A (c)), the separate identification of TV2 was not possible. In the present study, we also searched for the three alternative splice forms previously described in one patient with IMO [16]. A splice form with retention of intron 19 was verified within others indicated by the dbEST search, as described in Table 1. The form lacking exon 19 (Del_Ex19) (Fig 1B (b)) was detected by amplification with primers located in exons 18 and 20, and the form with extension of exon 18 to position +170 in authentic intron 18 and skipping of exon 19 (Part_Ret_Int18+Del_ex19) (Fig. 1B (d)) was detected by amplification with primers located in intron 18 and exon 20. The more highly expressed forms including exon 19 (Fig. 1B (a) and (c)) were depleted by digestion with NlaIII. Tissue distribution of the TCIRG1 splice forms Amplification of the region between exons 17 and 20, common for TV1 and TV2, in 44 samples from 28 human tissues (Table 2) demonstrated that the gene is ubiquitously expressed. We studied tissue distribution of the following nine TCIRG1 splice forms: TV1 ex1_7, CrypEx_Int1, Part_Ret_Int13, Ret_Int5, Ret_Int12, Ret_Int15, Ret_Int18, Del_Ex19, and Part_Ret_Int18 + Del_ex19. As shown in Table 2, 19 samples from 15 tissues demonstrated all of these nine splice forms, 13 samples demonstrated eight splice forms, and 12 less than eight splice forms. In some cases, samples from the same tissue showed different numbers of splice forms (Table 2). We later tested whether the number of detected splice forms is related to RNA quality. The mean 28S/18S rRNA ratio and the mean percentage of 28S and 18S rRNA area in our samples were 1.0 ± 0.6 and 15.6 ± 12.8, respectively. These two characteristics correlated well (r = 0.74, p < 0.0001). We defined the
Fig. 2. Relationship between RNA quality index (x) and number of TCIRG1 splice forms (y) detected in a sample. The RNA quality index is the sum of the 28S/18S rRNA ratio and the percentage of rRNA area of a sample, both expressed as percentages of the mean of our group.
RNA quality index as the sum of the 28S/18S rRNA ratio and the percentage of rRNA area of a sample, both expressed as percentages of the mean for the group (Fig. 2). We observed correlation between RNA quality index and the number of splice forms detected (r = 0.59, p < 0.001) (Fig. 2). Verification of the TCIRG1 splice forms in cytoplasmic RNA of PBMC Expression levels of the nine TCIRG1 splice forms were evaluated by semi-quantitative RT-PCR in both total and cytoplasmic RNA from 18 PBMC samples. Six splice forms, i.e., CrypEx_Int1, Ret_Int12, Ret_Int15, Del_Ex19, Part_Ret_Int18 + Del_ex19, and TV1 ex1_7, demonstrated similar (less than twofold difference) levels of expression in cytoplasmic and total RNA samples. The other three splice forms, Ret_Int5, Part_Ret_Int13, and Ret_Int18, were represented in cytoplasmic RNA at significantly lower levels (the median difference was more than 4, p < 0.001, as illustrated in Figs. 3A–C). Analysis of the open reading frame (ORF) The TV1 and TV2 translations initiate in exons 2 and 7 of the gene, respectively, and terminate in exon 20 [5].
Fig. 3. TCIRG1 splice forms with differential expression in cytoplasmic (C) and total (T) RNA: example of detection in one representative PBMC sample. (A) Splice form with retention of intron 5. (B) Splice form with partial retention of intron 13. (C) Splice form with retention of intron 18. (D) POLR2K as internal control.
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Fig. 4. Schematic presentation of the TCIRG1 isoform ‘‘a’’ topology. Arrows 1–5 indicate sites of protein truncation due to the appearance of the premature stop codon for retention of introns 12, 13, 15, 18 or exclusion of exon 19, respectively.
The three-dimensional topology of the corresponding protein isoforms has not yet been established experimentally. Different methods for the prediction of transmembrane helix topology based on hydrophobicity analysis of the protein sequence predict 7 (ExPASy) to 12 (TopPred) transmembrane a-helices for isoform ‘‘a.’’ Based on the experimental topology model of a yeast TCIRG1 homolog Vph1p [22], the isoform ‘‘a’’ could present a transmembrane region with nine a-helices (Fig. 4). The splice event CrypEx_Int1 did not change the ORF. Ret_Int5 in the full-length transcript results in a product similar to both TV1 and TV2, and does not allow prediction of its translation start site. Ret_Int12, Part_Ret_Int13, Ret_Int15 or Ret_Int18 generates a premature stop codon within the sequence corresponding to the intron, and Del_Ex19, within exon 20. Therefore, these five splice events would lead to the production of shortened proteins containing 3, 3, 5, 7, and 7 transmembrane domains, respectively (Fig. 4).
Discussion In the present study, we searched for alternative splice events in TCIRG1 in different human tissues. We validated six new alternative splice events indicated by a search in dbEST: CrypEx_Int1, Ret_Int5, Ret_Int12, Part_Ret_Int13, Ret_Int15, and Ret_Int18. They were observed in most of the 28 human tissues studied. Three unconfirmed putative splice events were indicated by single ESTs, and the failure in detecting them may be related to the low abundance or low frequency of these forms. We also investigated tissue distribution of the previously reported TCIRG1 splice forms. First, we verified the tissue distribution of TV1. The previous data about its expression were controversial: Li et al. [6], showed osteoclast-specific expression of the TV1 by Northern blot, and this finding was cited by several authors [7,10,16]; however, Scott and Chapman [23] showed ubiquitous distribution of TV1 by RT-PCR. Using RT-PCR, we also observed the product TV1 ex1_7 in
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most tissues. The difference between the results obtained by RT-PCR and Northern blot could be explained by the lower sensitivity of the latter. The data about almost exclusive expression of TV2 in immune tissues were also obtained by Northern blot [12], and, therefore, need further confirmation. Unfortunately, we were not able to design a strategy for specific amplification of this variant due to its high similarity to the other longer transcript (Fig. 1A). Next, we studied three TCIRG1 splice events, namely Ret_Int19, Del_Ex19, and Part_Ret_Int18 + Del_ex19, described as aberrant, related to IMO [16]. Although Ret_Int19 was not detected in any of our samples, the two other splice events were observed by us in various tissues of healthy individuals. Thus, we studied the nine alternative splice forms of the TCIRG1 gene and demonstrated that they occur in most tissues. However, some of the splice forms were not detected in a number of samples. The RNA quality in our samples varied strongly since it depends on tissue type [24,25] and RNA isolation method [26]. We observed that the number of splice events detected in our samples correlated with the RNA degradation level and therefore it was not possible to perform a precise analysis of their tissue-specific distribution. However, the expression analysis suggests that tissue distribution of the TCIRG1 splice forms is nearly ubiquitous. To verify whether TCIRG1 transcripts subjected to these alternative splice events could be potentially translated, we searched for the TCIRG1 splice forms in cytoplasmic RNA of PBMC samples. Six splice forms demonstrated similar levels of expression in cytoplasmic and total RNA, and therefore they are efficiently exported from the nucleus and could not be splicing intermediates. Three splice forms—with Ret_Int5, Part_Ret_Int13, and Ret_Int18—were hardly detected in cytoplasmic RNA. Although forms with simple intron retention could represent splicing intermediates, Part_Ret_Int13 uses an alternative acceptor splice site and, therefore, represents a bona fide alternative splice event. The first possible explanation for a low presentation of a splice form in cytoplasm is depletion by nonsense-mediated mRNA decay [27]. However, not all TCIRG1 splice forms generating premature stop codons are depleted. The second explanation could be regulation at the level of the nuclear export [28]. Recently, differential nucleocytoplasmic distribution of splice variants was also demonstrated for the human gene ADAM33 [29], but the relationship between alternative splicing pathways, RNA degradation, and nuclear export has not yet been studied. Also, it has been recently reported that nucleocytoplasmic distribution of mRNA could be regulated in a tissuespecific manner [30]. Therefore, the nucleocytoplasmic distribution of TCIRG1 splice forms warrants studies in other tissues.
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Our data indicate that splicing in TCIRG1 is extremely complex. Amplifications with primers specific for different alternative splice events yielded multiple bands, suggesting that alternative splice events in the gene may occur in various combinations (data not shown). The maximum number of possible combinations taking into account the nine splice types is 128. However, CrypEx_Int1 and Ret_Int5 did not modify the protein, and all the other alternative splice events identified in this study led to the appearance of the premature stop codon near the splice event. Thus, the amino acid sequence coded by a transcript containing multiple alternative splice events between exons 12 and 20 depends only on the first event. In summary, we validated six new alternative splice events in the TCIRG1 gene in various human tissues. We demonstrated that transcripts using the TV1 transcription start site and two splice forms previously described in one patient with infantile malignant osteopetrosis are also expressed in various tissues of healthy individuals. Studies of these nine TCIRG1 splice forms in cytoplasmic RNA of PBMC samples showed that at least six of them could be efficiently exported from the nucleus. Since various products with nearly ubiquitous tissue distribution are generated from TCIRG1, this gene may be involved in other processes besides immune response and bone resorption.
[6]
[7]
[8]
[9]
[10]
[11]
Acknowledgments We thank Dr. Emmanuel Dias Neto for helpful discussion of the manuscript and Dr. Paulo S.L. de Oliveira for help with the prediction of protein structure. We are grateful to Drs. Davimar M. Borducchi, Silvia Daher, and Mario Dolnikoff for help with sample collection. We thank Dr. Rui M.B. Maciel and Gustavo S. Guimara˜es for help with the DNA sequencing. This research was supported by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP) and Conselho Nacional de Pesquisa (CNPq). A.S. Smirnova is the recipient of a FAPESP fellowship.
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