Regulation of TMEM16A altrenatice splincing

TMEM16A/Anoctamin1 is a novel calcium-\uad\u2010activated chloride channel involved in neuronal and cardiac excitation, vascular tone, pain perception and olfactory and sensory signal transduction and GI tract motility. It is also associated to diverse type of cancer including breast cancer malignancy. Alternative splicing (AS) of exons 6b, 13 and 15 generates functionally distinct TMEM16A isoforms with different electrophysiological properties. To study their splicing regulation, I performed in minigene system a systematic analysis of exonic and intronic regulatory elements followed by co-\uad\u2010transfection of a panel of splicing regulatory factors. Analysis of TMEM16A pre-\uad\u2010mRNA splicing supports a model in which each exon is regulated by different cis-\uad\u2010 and trans-\uad\u2010acting elements. Exon 6b inclusion is regulated primarily by SRSF9 and TRA2B, through a unique GAA-\uad\u2010rich ESE element. Exon 15 is enhanced only by TIA1 and FOX1 and this effect is mediated by downstream intronic sequences. On the other hand, the small exon 13, included in most human tissues, was mainly skipped in the minigene and only FOX1 and U2AF65 enhanced its inclusion. To understand if there is any preferential association between three AS exons, I have evaluated TMEM16A isoforms using a long range RT-\uad\u2010PCR assay that amplifies transcripts across the AS events. Coordination between distant alternative spliced exons in the same gene has been suggested to be an important mechanism to regulate gene expression but very few genes have been studied in detail. I observed that the selection of exons 6b and 15 is preferentially coordinated in several human normal tissues: mature transcripts that predominantly include exon 6b tend to exclude exon 15. Unexpectedly, this coordination was not conserved in mouse tissues. This was mainly due to the fact that exon 15 was largely and predominantly excluded in the mouse, a fact that suggest a peculiar evolutionary conservation of AS in this gene. To explore if changes in splicing coordination of the two major AS events are associated to cancer development I evaluated normal mammalian tissue and corresponding breast tumors of the same cohort, obtained from surgical excision (n=18). The distribution of individual AS events did not change between normal and tumor tissues. However, the TMEM16A splicing coordination increased significantly in tumors. Indeed, the splicing coordination was present in 50% of normal mammalian breast tissues and in 84% in tumors. In conclusion this study identifies several cis-\uad\u2010acting elements and trans-\uad\u2010acting factors involved in the regulation of TMEM16A Alternative Splicing and provides evidence of its intragenic splicing coordination. The increase of TMEM16A splicing coordination observed in breast tumor, might represent a common event in genes with multiple AS events

Regulation of TMEM16A Chloride Channel Properties by Alternative Splicing*

Expression of TMEM16A protein is associated with the activity of Ca2+-activated Cl− channels. TMEM16A primary transcript undergoes alternative splicing. thus resulting in the generation of multiple isoforms. We have determined the pattern of splicing and assessed the functional properties of the corresponding TMEM16A variants. We found three alternative exons, 6b, 13, and 15, coding for segments of 22, 4, and 26 amino acids, respectively, which are differently spliced in human organs. By patch clamp experiments on transfected cells, we found that skipping of exon 6b changes the Ca2+ sensitivity by nearly 4-fold, resulting in Cl− currents requiring lower Ca2+ concentrations to be activated. At the membrane potential of 80 mV, the apparent half-effective concentration decreases from 350 to 90 nm when the segment corresponding to exon 6b is excluded. Skipping of exon 13 instead strongly reduces the characteristic time-dependent activation observed for Ca2+-activated Cl− channels at positive membrane potentials. This effect was also obtained by deleting only the second pair of amino acids corresponding to exon 13. Alternative splicing appears as an important mechanism to regulate the voltage and Ca2+ dependence of the TMEM16A-dependent Cl− channels in a tissue-specific manner