The Alternative Splicing Of Cdh23 Exon 68: Biological Function And Regulatory Mechanism

Sensory hair cells are required for hearing and balance functions in animals.On the apical surface of hair cell,there are many actin-filled stereocilia and a single microtubulecontaining kinocilium.In mature hair cells,the hair bundle is formed of stereocilia aligned in rows of graded heights and the mechanoelectrical transduction(MET)channels are located at the top of the two shorter rows of stereocilia.Opening of the MET channels is regulated by tip links which connects the sidewall of the taller row of stereocilia to the top of the adjacent lower stereocilia.The deflection of the hair bundle toward the taller row increases tension in tip links and thus opens MET channels.Conversely,the deflection toward the opposite direction closes MET channels.Tip links are composed of CDH23 and PCDH15,with CDH23 at the upper end and PCDH15 at the lower end.Both proteins are atypical cadherins and their extracellular segment is calcium-dependent to form stable heteropolymers.Regulated by different transcription initiation sites,CDH23 expresses three isoforms.The longest V1 isoform,which constitutes tip link,consists of 27 cadherin repeat domains in extracellular segment.The V2 isoform consists of 7 cadherin repeat domains in extracellular segment and the shortest V3 isoform is a cytosolic protein.In mouse,Cdh23 gene contains 69 exons,of which exon 68 is 105 bases long and encodes 35 amino acids in the intracellular segment.Exon 68 is regulated by tissue-specific alternative splicing and Cdh23(+68)is only detected in the inner ear so far.The biological function and mechanism of this alternative splicing regulation have not been clear.To examine the biological role of exon 68 splicing,we established knockout mice with Cdh23 exon 68 deleted.We found that Cdh23 exon 68 knockout leads to age-related deafness and noise-induced deafness.In the early stage of hair cell development,tip links can still form and function in the knockout mice.After hair cell maturation or noise stimulation,the number of tip links in the knockout mice was reduced,accompanied by decreased MET channel function.These results indicate that CDH23(+68)is necessary for the stability of tip links.We further found that CDH23-V3 isoform can localize to the stereocilia and the peptide encoded by exon 68 mediated intracellular interaction between V1(+68)and V3(+68),which is compromised by certain deafness-associated mutations in exon 68.In summary,we consider that CDH23-V1(+68)and CDH23-V3(+68)are polymerized by intracellular interaction,which is necessary for the stability of tip links.Although the tip links can still form after the absence of exon 68,the CDH23(-68)-formed tip links are less stable than regular CDH23(+68)-formed tip links,which eventually contributes to age-related or noiseinduced hearing loss.In the second part of this study,in order to understand the mechanisms of the alternative splicing regulation of Cdh23 exon 68,we constructed reporter gene for exogenous expression to screen important splicing regulatory proteins known to be expressed in the inner ear.We found that RBM24 and RBM38 can promote the inclusion of exon 68.RBM24 is an RNAbinding protein specifically expressed in the hair cells in inner ear.Inclusion of exon 68 was significantly reduced in both Rbm24 knockdown cell lines and Rbm24 knockout mouse inner ears.Furthermore,we found that PTBP1 can inhibit the inclusion of exon 68 and participated in the regulation of alternative splicing of Cdh23 exon 68 together with RBM24 and RBM38.CDH23 is an important deafness-related gene.This study stimulates the understanding of the function of CDH23 at tip links of hair cells,and reveals the regulatory mechanism of alternative splicing of exon 68.This study provides theory instruction and scientific basis for the pathogenesis,diagnosis and treatment of age-related deafness and noise-induced deafness caused by mutation in exon 68 of CDH23 gene.