Increased complexity of Tmem16a/Anoctamin 1 transcript alternative splicing

Kate E O'Driscoll 0 Rachel A Pipe 0 Fiona C Britton 0 0 Department of Physiology and Cell Biology , 1664 North Virginia Street , University of Nevada School of Medicine , Reno, Nevada 89557-0046 , USA Background: TMEM16A (Anoctamin 1; ANO1) is an eight transmembrane protein that functions as a calciumactivated 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. Results: 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. Conclusion: 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. - Background Alternative splicing of pre-mRNAs is a powerful regulatory mechanism that can increase mRNA transcript variety and effect functional diversification of proteins [1]. 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 [9], 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 [19]. 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 [13]. 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 [22]. 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 [22]. Exon 13, encoding 4 amino acids, contributes significantly to TMEM16A channel kinetics, since exclusion of this exon significantly reduces the voltage dependence of activation [22]. A recent study showed that exon 15 (encoding 26 amino acids) exclusion results in significantly faster activation and deactivation kinetics [23]. 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 [23]. 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 [24] and vascular [25] 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. Methods 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 g 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 95C for 10 min, then 35 cycles of 95C for 15 sec, Ta for 20 sec and 72C for 30-60 sec, followed by a final step at 72C 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 (...truncated)