Increased complexity of Tmem16a/Anoctamin 1 transcript alternative splicing
© O'Driscoll et al; licensee BioMed Central Ltd. 2011
Received: 23 March 2011
Accepted: 8 August 2011
Published: 8 August 2011
TMEM16A (Anoctamin 1; ANO1) is an eight transmembrane protein that functions as a calcium-activated chloride channel. TMEM16A in human exhibits alternatively spliced exons (6b, 13 and 15), which confer important roles in the regulation of channel function. Mouse Tmem16a is reported to consist of 25 exons that code for a 956 amino acid protein. In this study our aim was to provide details of mouse Tmem16a genomic structure and to investigate if Tmem16a transcript undergoes alternative splicing to generate channel diversity.
We identified Tmem16a transcript variants consisting of alternative exons 6b, 10, 13, 14, 15 and 18. Our findings indicate that many of these exons are expressed in various combinations and that these splicing events are mostly conserved between mouse and human. In addition, we confirmed the expression of these exon variants in other mouse tissues. Additional splicing events were identified including a novel conserved exon 13b, tandem splice sites of exon 1 and 21 and two intron retention events.
Our results suggest that Tmem16a gene is significantly more complex than previously described. The complexity is especially evident in the region spanning exons 6 through 16 where a number of the alternative splicing events are thought to affect calcium sensitivity, voltage dependence and the kinetics of activation and deactivation of this calcium-activated chloride channel. The identification of multiple Tmem16a splice variants suggests that alternative splicing is an exquisite mechanism that operates to diversify TMEM16A channel function in both physiological and pathophysiological conditions.
Alternative splicing of pre-mRNAs is a powerful regulatory mechanism that can increase mRNA transcript variety and effect functional diversification of proteins . Within the cardiovascular system, alternative splicing affects cardiac function by regulating proteins involved in cellular excitation, including ion channels [2–8].
Calcium-activated chloride currents have been recorded in cardiac muscle cells from various species including mouse , and play an important role in the cardiac action potential [10–12]. In 2008, three independent groups identified Tmem16a as a strong candidate gene to encode (or at least a major component of) a calcium-activated chloride channel [13–15]. Tmem16a belongs to a family of ten mammalian paralogs (Tmem16 (a-h, j-k)) that are highly conserved membrane spanning proteins. In recombinant expression systems, Tmem16a (or Ano1) and Tmem16b (or Ano2) generate calcium-activated chloride currents [13–18] with similar biophysical and pharmacological properties to currents recorded from native tissues . We and others have identified Tmem16a expression in mouse and human heart [20, 21].
The human TMEM16A gene exhibits three alternatively spliced exons (6b, 13 and 15) as well as an alternative transcription start site . Ferrera et al. reported that the biophysical properties of human TMEM16A are regulated by alternative splicing and TMEM16A splice variants form functional channels that display different properties . The alternative exon 6b (encoding 22 amino acids) may play an important role in the regulation of the TMEM16A channel by calcium, since exclusion of this exon increases the calcium sensitivity of the channel ~ 4-fold . Exon 13, encoding 4 amino acids, contributes significantly to TMEM16A channel kinetics, since exclusion of this exon significantly reduces the voltage dependence of activation . A recent study showed that exon 15 (encoding 26 amino acids) exclusion results in significantly faster activation and deactivation kinetics . In addition, Mazzone et al, showed significant differences in expression of alternatively spliced TMEM16A exons in patients with diabetic gastroparesis when compared to non-diabetic controls . Therefore, it appears that alternative splicing of human TMEM16A plays an important role in the regulation of calcium-activated chloride channel function.
The reported mouse Tmem16a gene [GenBank: NC_ 000073] is composed of 25 exons that code for a 956 amino acid protein [GenBank: NP_848757]. Unlike human, mouse Tmem16a, as annotated, does not contain alternative exons 6b, 13 or 15. We and others however, have reported that Tmem16a transcripts containing these alternative exons are expressed in mouse stomach, intestine  and vascular  smooth muscle tissues. It is likely that alternative splicing of Tmem16a transcript in mouse may lead to a number of different TMEM16A channel proteins with altered biophysical properties similar to human TMEM16A [22, 23]. In this study our aim was to provide detailed information of the structure of the mouse Tmem16a gene and to investigate if Tmem16a transcript undergoes alternative splicing to generate channel diversity in mouse heart. This study demonstrates that the structure of Tmem16a gene is significantly more complex than previously indicated. The complexity is especially evident in the region containing exons 6 through 16. Determining the variation of Tmem16a transcript expression in heart is an important foundation for future studies of the physiological role of Tmem16a channels in heart.
RNA isolation and RT-PCR
Total RNA was isolated from mouse tissues using TRIzol reagent (Invitrogen, Carlsbad, CA). Human heart RNA was purchased from Agilent Technologies (Santa Clara, CA). First-strand cDNA was prepared from 1 μg of RNA using oligo(dT)(12-18) primer and Superscript II reverse transcriptase (Invitrogen). AmpliTaq Gold® PCR reagent (Applied Biosystems, Foster City, CA) was used to amplify each of the Tmem16 paralogs and Tmem16a splice variants. PCR primers were designed using the mouse and human Tmem16a mRNA sequences [GenBank: NM_178642 and GenBank: NM_018043]. Details of the primer sets used are provided in additional file 1, Table S1 and Table S2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control for cDNA integrity. No template PCR reactions served as controls for primer contamination. PCRs were performed in a 2720 Thermal Cycler (Applied Biosystems). Amplification consisted of 95°C for 10 min, then 35 cycles of 95°C for 15 sec, Ta for 20 sec and 72°C for 30-60 sec, followed by a final step at 72°C for 7 min.
Splice variant identification, sequencing and bioinformatics
PCR products were resolved on 2-3% super fine agarose (Amresco, Solon, OH) gels along with a 100 bp molecular weight marker. Tmem16a amplification products were either purified (QIAquick Gel Extraction Kit, Qiagen, Valencia, CA) or TA cloned into the pcDNA3.1 vector (Invitrogen). All fragment sequencing was performed at the Nevada Genomics Center. Nucleotide and protein sequences were analyzed using Vector NTI version 11 software (Invitrogen) and BLASTN software at the National Center for Biotechnology Information (NCBI) database http://www.ncbi.nlm.nih.gov/BLAST/.
Transmembrane regions were predicted using the CBS TMHMM Server v 2.0 http://www.cbs.dtu.dk/services/TMHMM/ and TMpred http://www.ch.embnet.org/software/TMPRED_form.html programs. The TAndem Splice Site DataBase (TassDB2) http://gen100.imb-jena.de/TassDB2/ was used to confirm alternative tandem splice sites in mouse and human Tmem16a. The Scansite database http://scansite.mit.edu/motifscan_id.phtml was used to identify motifs in the TMEM16A alternative splice variants.
Results and Discussion
Tmem16a genomic structure is highly conserved in mouse and human
Genomic Organization of Tmem16a
3' Acceptor and 5' Donor splice site sequences
CAGGTG gt agga
ttgcag ACGCCA---------------------GGCACG gt gagt
tcacag TTGCTG---------------------GAGGAT gt gagt
ctctag ACCAAA---------------------AAGAAG gt gagt
ccacag GTGTAC----------------------ACACCT gt gagt
ctgcag ATTCGA----------------------ACAATA gt gagt
ccctag GTCTAT---------------------GCATGG gt aagg
aaacag GGCAAG--------------------AATACG gt aaga
ttaaag GTATCA----------------------CACGAT gt aagt
tcacag GGGGAC--------------------AGGAAA gt aagt
ccccag CTCCTG----------------------GGTCAG gt aagt
gcatag GAAATA---------------------CCCCAG gt aggc
ttacag TATGGA----------------------TCTGGG gt aagt
ctacag CTGCCA---------------------GAGGAG gt gagt
ccgcag GAAGCT---------------------GTCAAG gt ttga
ttccag CACCTT----------------------CAGAGG gt cagt
tctcag GATCAT---------------------AAAGAG gt acag
cctcag AAGTGC----------------------AAATTG gt actt
ttgcag ACCGAC----------------------TTCATG gt aagt
ctgcag ATCGCA----------------------AGATTG gt gagt
cctcag AGGTCC---------------------AGGCCG gt agga
tcccag GTTTGT---------------------GAGGAG gt aata
ctacag TGTGCC----------------------CATCCC gt gagt
ctgcag GAAGAT---------------------AAATGA gt gagt
ctgcag TCATTC-----------------------ACATCG gt aagt
ctctag GCATCT-----------------------ATTAAT gt aagt
tggcag GCCTTT----------------------CTGCAG gt acta
ttgcag GTATAA-----------------------TTCCAG gt atgt
Identification of Tmem16a splice variant expression in mouse heart
A comparison of the amino acid sequences of mouse and human alternative exons show a high similarity; the four amino acids (EAVK) of exon 13 are 100% identical, the 22 and 26 amino acids encoded by exon 6b and exon 15, share 95.5% and 80.8% identity, respectively. Considering the high percentage amino acid identity of these exon between the two species, we can expect that the regulatory properties attributed to these channel domains in human [22, 23], will most likely confer similar functional roles in the mouse, in that, exon 6b may confer calcium sensitivity, exon 13 may contribute to voltage dependence and exon 15 may play a role in the activation and deactivation kinetics of this calcium activated chloride channel. The inclusion of alternative exons 6b, 13 and 15 would result in additional amino acids in intracellular channel domains and are not predicted to alter the eight transmembrane topology of the TMEM16A protein.
Tmem16a transcripts with combinations of alternative exons 6b, 13 and 15 create diversity
Both mouse and human heart express the following three exon combinations: Tmem16a (-6b, +13, -15), Tmem16a (+6b, +13, -15), and Tmem16a (-6b, +13, +15). We did not identify Tmem16a transcripts consisting of exon 6b alone (+6b, -13, -15) or a Tmem16a variant containing 6b and 15 but lacking exon 13 (+6b, -13, +15) in either mouse or human heart. It appears, at least in heart, that exon 6b is only expressed in the presence of exon 13. However, we did identify Tmem16a transcripts containing exon 15 in the absence of both exons 6b and 13 (-6b, -13, +15) in mouse heart. Interestingly, a Tmem16a transcript missing all three exons (-6b, -13, -15) was only identified in mouse heart, in agreement with the reported Tmem16a [GenBank: NM_178642] and although a human transcript variant 2 (-6b, -13, -15) [GenBank: NR_030691] exists we did not confirm its presence in human heart. All human TMEM16A transcripts contained exon 13, in agreement with [GenBank: NM_018043] (-6b, +13, +15). The largest Tmem16a variant consisting of all three exons was confirmed by sequence analysis in human heart. Although this Tmem16a variant was not confirmed in our screening of mouse heart clones, we cannot rule out its existence since our gel analysis indicates a band close to the expected size of this variant, albeit much weaker than in human (Figure 2B). Indeed, it is possible that different mouse and human tissues may express different combinations of these alternative exons, however, quantification of our data would not provide more information since the tissues examined in this study contain a mixed population of cells. The deduced amino acid sequence of mouse TMEM16A containing alternative exons combinations (6b, 13 and 15) all read in-frame, indicating that these alternative exons are additional channel components similar to those of human TMEM16A . BLASTn searches of dbEST and Refseq RNA databases identified a number of mouse ESTs containing exon 6b and exon 13. Indeed, one mouse EST [GenBank: BI151426] was identified for the +6b, +13, -15 exon combination. EST supporting query sequences were identified for mouse [GenBank: BI685387] containing exon 13 alone (-6b, +13, -15). No ESTs were found in mouse containing both exon 13 and 15, however this is the exon combination in human that corresponds to TMEM16A [GenBank: NM_018043].
In addition to alternative exons 6b, 13 and 15, we also identified a number of novel Tmem16a splicing events in mouse and human heart; exon 10 and exon 14 exclusion, the inclusion of a novel exon, and two intron retention events.
Novel Tmem16a transcripts with exon 10 or exon 14 exclusion
Sequence analysis revealed that the Tmem16a (-14) variant included exons 6b and 13 and excluded exon 15 (Figure 3B). Exclusion of exon 14 (72 bp) in Tmem16a RNA would result in an in-frame deletion of 24 amino acids in the first intracellular loop of the TMEM16A channel. A PKC site is present in exon 14 at position S462 [GenBank: NP_848757] and one could speculate that Tmem16a transcripts lacking exon 14 may encode channels that differ in modulation by PKC. Likewise, the expression of this variant was confirmed in mouse and human heart using primers which specifically amplify Tmem16a (-14) variants.
Tmem16a transcripts with a novel conserved exon 13b
Considering that the exon structures of Tmem16a are highly conserved amongst homologs, we investigated if similar novel exons could be identified in other species. A close examination of the genomic Tmem16a sequence from rat, human and rhesus monkey [GenBank: NC_005100, GenBank: NC_000011, GenBank: NC_007871,] revealed that similar sequences to mouse Tmem16a exon 13b, bordered by conserved splice sites could be identified. An alignment of the corresponding amino acid sequence of these four species (Figure 4B) demonstrates 97.6% overall consensus with an identity of 41.5%. Specific primers designed to amplify this novel exon in human heart were unable to identify its expression. Whether or not this exon is present in other human tissue types is unknown.
The expression of the novel exon 13b in mouse heart was investigated further using primer sets to specifically amplify the 5' and 3' regions surrounding the 120 bp exon (Figure 4C). Sequence analysis revealed some interesting observations. First, we confirmed that exon 13b is utilized in mouse heart. Secondly, exon 13b is present in several Tmem16a transcripts that differ in whether they also include or exclude alternative exons 13 or 15. Third, retention of the introns surrounding exon 13b increases the complexity of splicing in this region of Tmem16a transcript. RT-PCR with the primer set 5'Exon13b, in which primers bind within exon 11 and 13b produced two amplification products (Figure 4C). Sequencing analysis indicated one Tmem16a fragment consisted of exons 11, 12 and 13b and the other fragment consisted of exons 11, 12, 13, 13b plus the entire 161 bp intronic sequence between exon 13 and 13b (Figure 4C). The 3'Exon13b primer set, in which the primers hybridize across exon 12/13b junction and within exon 16, yielded three Tmem16a transcript fragments (Figure 4C). Two fragments differ in exon 15 inclusion/exclusion exons 12, 13b, 14, ± 15, 16, and an unexpected fragment consisting of exon 12, 13b, 14, -15, 16 plus the entire intronic sequence (273 bp) between exon 13b and 14 (Figure 4C). Intron retention events at the 5' and 3' of exon 13b are certainly the result of alternative Tmem16a splicing and not genomic contamination since the 5'Exon13b and 3'Exon13b primers span at least 14.4 kb and 4.4 kb of genomic sequence, respectively.
The intron retention events at the 5'and 3' of exon 13b would introduce a premature stop codon in the Tmem16a transcript, resulting in a truncated protein just after the "EAVK" amino acids encoded by exon 13. The complete retention of an intron in a mature transcript is one of the least expected forms of alternative splicing and may occur due to weak splice sites, cis-regulatory elements or short intron lengths . Indeed, the introns between exons 13 and 13b and between exon 13b and 14 are two of the shortest in the Tmem16a genomic sequence (see Table 1). Intron retention can result in the insertion of a premature stop codon being introduced to the mature transcript which may then be degraded by nonsense mediated decay . Indeed, decreasing the mRNA levels of certain genes can be the purpose of alternative splicing . However, sometimes the retention of an intron in the coding region can have a biological effect. For example, an intron retaining isoform of the KCNMA1 gene, which encodes the large conductance calcium-activated potassium channel (BK), facilitates splice variant regulation which contributes to structural and functional diversity of BK channel proteins in hippocampal neurons [33, 34].
Tmem16a transcript scanning assay reveals additional novel splice variants
Mouse Tmem16a transcript scanning assay
sequences (5'→ 3')
Δ3' Exon 1
+ Exon 6b
+ Exon 13
+ Exon 15
+ Exons 6b + 13
+ Exons 13 + 15
+ Exon 13b
- Exons 10 +13 +15
- Exons 6b +13 -14
+ Exon 13
Exons 13 + 15
- Exon 18
Δ3' Exon 21
Human TMEM16A transcript scanning assay
sequences (5'→ 3')
+ Exon 6b
+ Exon 13
+ Exons 6b + 13
+ Exons 13 + 15
+ Exons 6b +13 + 15
- Exons 10 + 13
- Exons 10 + 13+15
+ Exons 6b + 13 - 14
- Exon 18
The Δ3'exon 1 and Δ3'exon 21 alternative donor sites are the result of wobble splicing occurring at donor splice sites located in close proximity, commonly known as tandem splice sites. Alternative splicing at tandem splice sites occurs frequently in many species and can result in subtle variations in transcripts and the proteins that they encode . Use of the Tmem16a Δ3'exon 1 splice site, results in the exclusion of the last 3 nucleotides (GTG) of exon 1. The mouse Tmem16a genomic sequence was queried against the TassDB database of alternative tandem splice sites , and the Δ3'exon 1 was recognized as tandem splicing at donor splice site with the motif GYNGYN. Indeed, a BLASTn search of the EST database revealed 27 ESTs without this 3' GTG and two ESTs that contained it. This alternative splice event is not expected to alter TMEM16A protein since exon 1 is non-coding. Skipping of exon 1 and part of exon 2 has been reported in human TMEM16A , however, Ferrera et al failed to identify transcripts lacking this region  and considered that its splicing may be under the control of an alternative promoter and influenced by certain physiological or pathophysiological conditions. Indeed, a recent study showed that a novel TMEM16A variant with an alternative 5' end is highly expressed in gastric smooth muscle of patients with diabetic gastroparesis compared with controls . Apart from Δ3'exon 1 tandem splice site, our study did not identify additional variation at the 5'end. The Δ3'exon 21 tandem splice site would result in 4 additional (GTGA) nucleotides at the 3' end of exon 21. This is predicted to shift the protein out of frame at amino acid M702 [GenBank: NP_848757], resulting in a truncated TMEM16A protein with an altered C-terminus of ~84 kDa. According to TassDB2  this tandem splice site is conserved in human, dog and chicken Tmem16a sequence.
Distribution of novel Tmem16a alternative splice variants in other tissues
Distribution of Tmem16a exon variants in mouse tissues
+ exon 6b
- exon 10
+ exon 13
+ exon 13b
- exon 14
+ exon 15
- exon 18
This report provides details of Tmem16a genomic organization and our analyses have detected previously unidentified alternative exons. We identified Tmem16a transcript variants consisting of different exon usage events involving exons 6b, 10, 13, 14, 15 and 18. Many of these exons are expressed in various combinations. A number of other splicing events including a novel exon 13b, tandem splice sites of exon 1 and 21 and two intron retention events were also identified. The alternative splicing events indentified here indicate that a varied population of TMEM16A channels is expressed in mouse heart and other tissues. The heart is a highly heterogeneous tissue composed of numerous cell types tailored for specialized function. It is possible that many Tmem16a variants can be co-expressed in the same cells, or differentially distributed in distinct cell types within the myocardium. Determining the localization of different Tmem16a isoforms within tissues has important implications with regard to the function of Tmem16a. The functional consequence of TMEM16A channel diversity remains to be determined. We can speculate however, that TMEM16A variants would create channels with altered biophysical properties, such as that reported for exon 6b, 13 and 15 in TMEM16A [22, 23]. It is also possible that regulation of the channels may be altered since several of these alternatively spliced exons contain phosphorylation sites and interacting domains, for example, PKC and CamKII (exon 18), PKA (exon 6b), Erk-D domain (exon 10) and a Src-SH3 domain (exon 15). TMEM16A channels have been shown to form homodimers [40, 41], therefore, it is likely that TMEM16A variants may interact with each other to form functional channels in a similar manner to that reported for the hERG family . Another possibility is that alternatively spliced isoforms may associate with additional accessory proteins. It is evident from this study that alternative splicing is an exquisite mechanism that operates to diversify TMEM16A channel structures by the combinatorial selection of alternatively spliced events. Future recombinant expression of different Tmem16a variants will reveal their physiological role.
Acknowledgements and Funding
The authors would also like to acknowledge Carly Gertler for excellent technical assistance. This project was supported by a NIH grant 1RO1HL091238 to FCB.
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