Alternative splicing of inner-ear-expressed genes

Abstract

Alternative splicing plays a fundamental role in the development and physiological function of the inner ear. Inner-ear-specific gene splicing is necessary to establish the identity and maintain the function of the inner ear. For example, exon 68 of Cadherin 23 (Cdh23) gene is subject to inner-ear-specific alternative splicing, and as a result, Cdh23(+68) is only expressed in inner ear hair cells. Alternative splicing along the tonotopic axis of the cochlea contributes to frequency tuning, particularly in lower vertebrates, such as chickens and turtles. Differential splicing of Kcnma1, which encodes for the α subunit of the Ca²⁺-activated K⁺ channel (BK channel), has been suggested to affect the channel gating properties and is important for frequency tuning. Consequently, deficits in alternative splicing have been shown to cause hearing loss, as we can observe in Bronx Waltzer (bv) mice and Sfswap mutant mice. Despite the advances in this field, the regulation of alternative splicing in the inner ear remains elusive. Further investigation is also needed to clarify the mechanism of hearing loss caused by alternative splicing deficits.

Keywords

alternative splicing / inner ear / hearing loss / hair cells

Introduction

Alternative splicing is an important process employed to modulate the function of eukaryotic proteins and is an important contributor to proteomic diversity. During alternative splicing, the exons of precursor mRNAs (pre-mRNAs) are spliced together in different arrangements to produce distinct mature mRNAs, which ultimately results in structurally and functionally distinct protein variants [ 1]. Approximately 95% of human multi-exon genes undergo alternative splicing [ 2]. This process is particularly common for genes that are expressed in the nervous system [ 3]. Alternative splicing affects neuronal development in many aspects, including cell fate determination, axon guidance, and synaptogenesis [ 4, 5]. Alternative splicing errors contribute to neurological and neuromuscular diseases, such as spinal muscular atrophy and frontotemporal dementia [ 6, 7].

The inner ear is an important part of the peripheral nervous system and is responsible for reception and transduction of sound and balancing information. Many inner-ear-expressed genes undergo alternative splicing, and errors in this process result in syndromic or non-syndromic hearing loss. For example, a mutation in the gene encoding for splicing factor Ser/Arg repetitive matrix 4 (SRRM4) causes alternative splicing aberrance and hearing loss in mice [ 8]. Another example comes from the finding that a missense mutation in MYO7A gene causes reinforcement of a minor alternative splicing event and results in a mild form of retinopathy and deafness [ 9]. Meanwhile, several deafness-causing mutations are localized to alternative exons in hearing-related genes, such as MARVELD2, MYO15A, and USH1C [ 10– 13]. The existence of multiple alternative splice variants in certain hearing-related genes may help to explain why different mutations in these genes result in substantial variation in clinical phenotypes.

In this review, we will focus on the role of alternative splicing in the inner ear. We will start with a discussion of inner-ear-specific gene splicing. We will also discuss the role of alternative splicing in frequency tuning. Then, we will focus on the splicing factors that have been shown to play important roles in the inner ear. We will see that although substantive advances have been achieved, the regulation of alternative splicing in the inner ear remains largely unknown. Further investigation is also needed to clarify the mechanism of hearing loss caused by alternative splicing deficits.

Inner-ear-specific gene splicing

Tissue-specific gene splicing is critical to establish tissue identity and maintain tissue function. For example, MEF2D is a ubiquitously expressed transcriptional factor, and during myogenesis, Mef2D mRNA alternatively uses mutually exclusive exons to produce a muscle-specific isoform, which shows altered transcriptional activity by changing its susceptibility to phosphorylation by protein kinase A (PKA) [ 14, 15]. Microarrays and deep sequencing have revealed more tissue-specific splicing patterns on a genome-wide scale [ 16, 17].

The most well-known inner-ear-specific gene splicing occurs in Cadherin 23 (Cdh23) gene, which encodes for the tip-link protein Cadherin 23 (CDH23) [ 18, 19] (Table 1). CDH23 mutations cause tip-link interruption and stereocilia disorganization, resulting in syndromic and non-syndromic hearing loss in humans and mice [ 20– 22]. Mouse Cdh23 gene contains 69 exons, and when exon 68 is subject to alternative splicing, two Cdh23 splicing variants, Cdh23(+68) and Cdh23(−68), are produced [ 23]. Notably, although Cdh23(−68) is ubiquitously expressed in many tissue types, Cdh23(+68) is only detected in the inner ear [ 18, 24, 25] (Fig. 1). Exon 68 is 105 base pairs long, encoding 35 amino acids in the cytoplasmic domain with no homology to other proteins. The inclusion of exon 68 has been suggested to affect the conformation as well as protein–protein interaction of CDH23 [ 24, 26, 27].

Until now, not much is known on how tissue-specific splicing patterns are established. Several splicing factors have been identified that are preferentially expressed in particular tissue types, such as NOVA (encoded by Nova) in the brain and ESRP1/2 in epithelial cells. These splicing factors are involved in tissue-specific splicing in these tissue types [ 28– 31]. Tissue-specific splicing can also be achieved by tissue-specifically expressed splicing regulators other than splicing factors, such as the alternative splicing repressors PTBP1 and PTBP2. PTBP1 is broadly expressed in nonneuronal cells, whereas PTBP2 is abundantly expressed in mature neurons. This tissue-specific distribution of PTBP1/PTBP2 plays a major role in tissue-specific alternative splicing [ 32, 33]. At present, such splicing factors or regulators have not been identified in the inner ear. In a microarray screening, approximately 50 splicing factors were identified to be expressed in chicken inner ear, none of which had yet been shown to be inner-ear-specific [ 34]. The identification of inner-ear-specific splicing factors or regulators is essential to understanding the mechanism of inner-ear-specific splicing.

Alternative splicing and frequency tonotopy

An amazing characteristic of the auditory system is its remarkable frequency selectivity and sensitivity over a large frequency range. For humans, the frequency ranges from 20 Hz to 20 kHz. Along the cochlear sensory epithelia, hair cells at each position are most sensitive to particular sound frequencies, resulting in the so-called frequency tonotopy. Many morphological and physiological properties contribute to this frequency tonotopy, including the physical properties of the basilar membrane and tectorial membrane, and hair cell stereocilia length, etc. [ 35]. Alternative splicing also plays an important role in this process, particularly in lower vertebrates, such as birds and reptiles.

In birds and reptiles, frequency tuning is achieved mainly through tonotopic differences in ion conductance within the hair cell membrane, which is mediated by the L-type voltage-gated Ca²⁺ channel (CaV_1.3 channel) and the Ca²⁺-activated K⁺ channel (BK channel) [ 36– 38]. The subunit composition and numbers of these channels determine the characteristic frequency of each hair cell. Approximately two decades ago, differential splicing of Kcnma1, which encodes for the a subunit of BK channel, was shown to affect the channel gating properties and is important for frequency tuning (Table 1). Chicken Kcnma1 is alternatively spliced along the tonotopic axis of the basilar papilla (Fig. 2), and its 11 alternative exons at 7 splice sites could generate up to 576 splicing variants [ 39, 40]. Different splicing variants generate kinetically distinct BK channels that contribute to frequency tuning [ 40, 41]. Similarly, turtle kcnma1gene is also alternatively spliced tonotopically along the basilar papilla, and different splicing variants generate kinetically different BK channels [ 42, 43].

In the mammalian cochlea, frequency tuning mainly depends on the mechanical properties of the basilar membrane, whereas alternative gene splicing appears to play a less significant role in frequency tuning. Nevertheless, tonotopic alternative splicing was present in certain genes, including Kcnma1. A total of 27 Kcnma1 splicing variants involving 7 alternative splicing sites were identified in mouse cochlea, and several of these splicing variants show different expression patterns along the tonotopic axis [ 44]. Kcnma1 knockout mice showed degenerated outer hair cells (OHCs) and developed progressive hearing loss [ 45]. Tonotopic splicing also occurs in Kcnq4, the gene encoding for a voltage-gated K⁺ channel whose mutations cause progressive hearing loss in humans and mice [ 46– 48] (Table 1). Beisel and colleagues showed that four Kcnq4 splicing variants are differently expressed along the tonotopic axis of the mouse cochlea [ 49]. The tonotopic splicing of these genes in the mammalian cochlea might play important roles in cochlear function.

At present, little is known about how tonotopic alternative splicing is regulated in the inner ear. In an effort to identify differentially expressed splicing activator(s) or repressor(s) responsible for tonotoic splicing along the chicken basilar papilla, a large-scale analysis of gene expression using a chicken genome array was performed. Although several genes showed different expression levels between the apex and base, no significant difference was observed in the expression of the splicing factors or repressors between these two regions [ 34]. Further investigation is needed to address this question.

Splicing factors in the inner ear

In spite of the failure in identifying inner-ear-specific splicing factors and tonotopically expressed splicing factors, researchers have identified several splicing factors that play important roles in hearing. Microarray analyses revealed approximately 50 splicing factors expressed in the chicken basilar papilla, many of which are involved in alternative splicing [ 34]. Noticeably, several splicing factors identified in this experiment belong to the SR protein family, including PNISR, SRRM1, SRSF3, SRSF5, SRSF6, SRSF10, TRA2A, and TRA2B. The precise expression patterns of these splicing factors in the inner ear are unknown. However, transcriptome analysis showed that these splicing factors are expressed in cochlear and vestibular hair cells as well as other cells in the inner ear (SHIELD; https://shield.hms.harvard.edu) [ 50, 51]. SR proteins share a serine/arginine-rich domain and play important roles in constitutive as well as alternative pre-mRNA splicing [ 52]. More splicing factors were believed to fall below the detection threshold of this analysis because of their low expression levels [ 34].

Until now, two splicing factors have been clearly shown to play important roles in hearing, both of which belong to the SR protein family. The first splicing factor is SRRM4, which contains three serine/arginine-rich domains (Fig. 3). Mutation of the Srrm4 gene causes alternative splicing defects and is responsible for profound hearing loss in Bronx Waltzer (bv) mice [ 8]. bv mice were originally identified in the late 1970s and are characterized by degenerated auditory and vestibular hair cells [ 53]. Notably, bv mice show inner hair cell (IHC) degeneration without OHC loss in the cochlea, and the OHCs of bv mice are functionally active [ 53– 57]. Further examination showed that IHCs are formed in the E17 bv mice but fail to form synapses with spiral ganglion neurons, resulting in degeneration by P3–P5 [ 58, 59]. A similar situation also occurs in vestibular hair cells [ 60].

The bv deafness gene was mapped to mouse chromosome 5 [ 61], and recently a 2710 bp deletion in the Srrm4 gene was identified to be responsible for the bv mouse phenotype [ 8] (Table 1). SRRM4, originally known as nSR100 (neural-specific SR-related protein of 100 kDa), regulates brain-specific alternative splicing of genes that function in neural cell differentiation [ 62]. The deletion of the Srrm4 gene in bv mouse removes a portion of the last intron and the entire coding region of the last exon, resulting in the disruption of SRRM4 protein expression. The alternative splicing of several genes are affected in the inner ear of bv mice, including Ap1s2, Cacnb2, Dtna, Dync1i2, Kif1b, Mef2d, Nrcam, and Phf21a [ 8]. In situ hybridization showed that, in the cochlea, Srrm4 is expressed specifically in IHCs, OHCs, and spiral ganglion neurons. In vestibular organs, Srrm4 is specifically expressed in hair cells. The specific expression of Srrm4 in cochlear and vestibular hair cells is supported by RNA sequencing results of fluorescence activated cell sorting (FACS)-sorted cells (SHIELD; https://shield.hms.harvard.edu) [ 50, 51]. Notably, although Srrm4 is abundantly expressed in other neuronal tissue types, such as cerebellum and neocortex, bv mice are subject to alternative splicing defects only in the inner ear, consistent with the fact that no phenotype exists in these tissue types in bv mice [ 8]. Further investigations are needed to discover why these tissue types and OHCs are unaffected by Srrm4 deficiency.

The second splicing factor that has been shown to play important roles in hearing is SFSWAP [ 63] (Table 1). SFSWAP, also known as SFRS8, contains a DRY_EERY domain and two SWAP domains as well as a serine/arginine-rich domain (Fig. 3). First identified in Drosophila, SFSWAP has been shown to regulate alternative splicing of several Drosophila genes, including Sfswap itself [ 64– 66]. In mammals, in vitro experiments indicated that SFSWAP regulates alternative splicing of Fibronectin and Ptprc [ 67– 69]. Recently, in a random lentiviral insertional mutagenesis study, Sfswap mutant mice were identified because of their significant circling behavior. The insertion of the lentiviral vector in intron 4 of Sfswap gene caused a significantly reduced expression level of Sfswap mRNA [ 63]. ABR measurement revealed that a moderate hearing threshold shift (20 dB to 25 dB) exists in Sfswap mutant mice.

In situ hybridization revealed that Sfswap is broadly expressed in the inner ear and brain since E10.5 [ 63]. In the cochlea, Sfswap expression is high in hair cells, supporting cells, and spiral ganglion cells but low in the surrounding tissue. In Sfswap mutant cochlea, regions that miss the third row of OHCs exist as well as regions that have ectopic IHCs. Meantime, given that mutant mice have a one third reduction in the length of the cochlea, the total numbers of IHCS and OHCs are significantly reduced [ 63]. Furthermore, loss of supporting cells was also observed in Sfswap mutant cochlea. Similar phenotypes have been observed previously in Jagged 1 (Jag1) heterozygous mice [ 70, 71], and consistently, Sfswap^−/−;Jag1^+/− mice show more severe phenotypes than expected from simple additive effects, indicating a genetic interaction between these two genes [ 63]. Jag1 is a well-known Notch ligand, and the inactivation of Jag1 in the developing inner ear leads to a severely disrupted organ of Corti [ 72, 73]. RT-PCR revealed that splicing of the Notch pathway genes is unaffected in the inner ear of Sfswap mutant mice. Thus, further investigation is needed to address the role of SFSWAP-regulated splicing in the inner ear.

Perspectives

We have discussed that alternative splicing is important for the development and physiological function of the inner ear and that alternative splicing deficits could cause hearing loss. In spite of the substantive advances in this field, the regulation of alternative splicing in the inner ear remains elusive. At the same time, further investigation is needed to understand the precise role of alternative splicing in hearing loss.

Furthermore, several other aspects of inner ear alternative gene splicing are worth noting, such as the regulation of alternative splicing by microRNAs (miRNAs). miRNAs are 21 to 25 nucleotide-long noncoding RNAs that regulate the expression of target genes at the posttranscriptional level. Alternative splicing factors/regulators have been shown to be targets of miRNA regulation. For example, Srsf1(SF2/ASF) mRNA is a target of miR-7, miR-10a, miR-10b, miR-28, and miR-505 [ 74– 76]. Another example involves PTBP1 and PTBP2, two repressors of alternative splicing that are expressed in nonneuronal cells and neurons, respectively, which have been mentioned previously [ 32, 33]. In neurons, miR-124 directly targets Ptbp1 mRNA and downregulates Ptbp1 expression [ 77]. In nonneuronal cells, such as myoblasts, miR-133 targets Ptbp2 mRNA and inhibits its translation [ 78]. In the inner ear, miRNAs might play similar regulatory roles toward splicing factors. In line with this, many miRNAs, including several ones mentioned previously, have been shown to be expressed in the inner ear and play important roles in hearing and balance [ 79– 82].

Ubiquitination has also been shown to regulate the expression of alternative splicing factors. Splicing factor SRSF1 is subject to ubiquitination and degradation by the proteasome in T cells [ 83]. Splicing factor KHDRBS3, also known as SLM2 or T-STAR, was shown to be targeted by E3 ubiquitin ligase SIAH1 for degradation [ 84]. Splicing factor RBM15 could be targeted by E3 ubiquitin ligase CNOT4 for degradation [ 85]. In the inner ear, several ubiquitination regulatory proteins, such as FBXO2 (OCP1), SKP1 (OCP2), SMURF1/2, and NEDD4, are present and important for the development and function of the inner ear [ 86– 89]. Whether these proteins regulate the degradation of alternative splicing factors in the inner ear awaits further investigation.

In conclusion, alternative splicing is important for inner ear development and function. More work is needed in the future to fully understand the mechanism how this process is regulated as well as to clarify why deficits of this process result in functional abnormality in the inner ear, such as hearing loss.

References

[1] Blencowe BJ. Alternative splicing: new insights from global analyses. Cell 2006; 126(1): 37–47

[2] Chen M, Manley JL. Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 2009; 10(11): 741–754

[3] Lee CJ, Irizarry K. Alternative splicing in the nervous system: an emerging source of diversity and regulation. Biol Psychiatry 2003; 54(8): 771–776

[4] Black DL, Grabowski PJ. Alternative pre-mRNA splicing and neuronal function. Prog Mol Subcell Biol 2003; 31: 187–216

[5] Li Q, Lee JA, Black DL. Neuronal regulation of alternative pre-mRNA splicing. Nat Rev Neurosci 2007; 8(11): 819–831

[6] Licatalosi DD, Darnell RB. Splicing regulation in neurologic disease. Neuron 2006; 52(1): 93–101

[7] Ranum LP, Cooper TA. RNA-mediated neuromuscular disorders. Annu Rev Neurosci 2006; 29(1): 259–277

[8] Nakano Y, Jahan I, Bonde G, Sun X, Hildebrand MS, Engelhardt JF, Smith RJH, Cornell RA, Fritzsch B, Bánfi B. A mutation in the Srrm4 gene causes alternative splicing defects and deafness in the Bronx waltzer mouse. PLoS Genet 2012; 8(10): e1002966

[9] Ben Rebeh I, Morinière M, Ayadi L, Benzina Z, Charfedine I, Feki J, Ayadi H, Ghorbel A, Baklouti F, Masmoudi S. Reinforcement of a minor alternative splicing event in MYO7A due to a missense mutation results in a mild form of retinopathy and deafness. Mol Vis 2010; 16: 1898–1906

[10] Ouyang XM, Xia XJ, Verpy E, Du LL, Pandya A, Petit C, Balkany T, Nance WE, Liu XZ. Mutations in the alternatively spliced exons of USH1C cause non-syndromic recessive deafness. Hum Genet 2002; 111(1): 26–30

[11] Riazuddin S, Ahmed ZM, Fanning AS, Lagziel A, Kitajiri S, Ramzan K, Khan SN, Chattaraj P, Friedman PL, Anderson JM, Belyantseva IA, Forge A, Riazuddin S, Friedman TB. Tricellulin is a tight-junction protein necessary for hearing. Am J Hum Genet 2006; 79(6): 1040–1051

[12] Nal N, Ahmed ZM, Erkal E, Alper OM, Lüleci G, Dinç O, Waryah AM, Ain Q, Tasneem S, Husnain T, Chattaraj P, Riazuddin S, Boger E, Ghosh M, Kabra M, Riazuddin S, Morell RJ, Friedman TB. Mutational spectrum of MYO15A: the large N-terminal extension of myosin XVA is required for hearing. Hum Mutat 2007; 28(10): 1014–1019

[13] Khateb S, Zelinger L, Ben-Yosef T, Merin S, Crystal-Shalit O, Gross M, Banin E, Sharon D. Exome sequencing identifies a founder frameshift mutation in an alternative exon of USH1C as the cause of autosomal recessive retinitis pigmentosa with late-onset hearing loss. PLoS ONE 2012; 7(12): e51566

[14] Martin JF, Miano JM, Hustad CM, Copeland NG, Jenkins NA, Olson ENA. A Mef2 gene that generates a muscle-specific isoform via alternative mRNA splicing. Mol Cell Biol 1994; 14(3): 1647–1656

[15] Sebastian S, Faralli H, Yao Z, Rakopoulos P, Palii C, Cao Y, Singh K, Liu QC, Chu A, Aziz A, Brand M, Tapscott SJ, Dilworth FJ. Tissue-specific splicing of a ubiquitously expressed transcription factor is essential for muscle differentiation. Genes Dev 2013; 27(11): 1247–1259

[16] Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB. Alternative isoform regulation in human tissue transcriptomes. Nature 2008; 456(7221): 470–476

[17] Pan Q, Shai O, Lee LJ, Frey BJ, Blencowe BJ. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nat Genet 2008; 40(12): 1413–1415

[18] Siemens J, Lillo C, Dumont RA, Reynolds A, Williams DS, Gillespie PG, Müller U. Cadherin 23 is a component of the tip link in hair-cell stereocilia. Nature 2004; 428(6986): 950–955

[19] Kazmierczak P, Sakaguchi H, Tokita J, Wilson-Kubalek EM, Milligan RA, Müller U, Kachar B. Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells. Nature 2007; 449(7158): 87–91

[20] Bork JM, Peters LM, Riazuddin S, Bernstein SL, Ahmed ZM, Ness SL, Polomeno R, Ramesh A, Schloss M, Srisailpathy CR, Wayne S, Bellman S, Desmukh D, Ahmed Z, Khan SN, Kaloustian VM, Li XC, Lalwani A, Riazuddin S, Bitner-Glindzicz M, Nance WE, Liu XZ, Wistow G, Smith RJ, Griffith AJ, Wilcox ER, Friedman TB, Morell RJ. Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherin-like gene CDH23. Am J Hum Genet 2001; 68(1): 26–37

[21] Di Palma F, Holme RH, Bryda EC, Belyantseva IA, Pellegrino R, Kachar B, Steel KP, Noben-Trauth K. Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D. Nat Genet 2001; 27(1): 103–107

[22] Bolz H, von Brederlow B, Ramírez A, Bryda EC, Kutsche K, Nothwang HG, Seeliger M, del C-Salcedó Cabrera M, Vila MC, Molina OP, Gal A, Kubisch C. Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D. Nat Genet 2001; 27(1): 108–112

[23] Di Palma F, Pellegrino R, Noben-Trauth K. Genomic structure, alternative splice forms and normal and mutant alleles of cadherin 23 (Cdh23). Gene 2001; 281(1-2): 31–41

[24] Siemens J, Kazmierczak P, Reynolds A, Sticker M, Littlewood-Evans A, Müller U. The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions. Proc Natl Acad Sci USA 2002; 99(23): 14946–14951

[25] Xu Z, Peng AW, Oshima K, Heller S. MAGI-1, a candidate stereociliary scaffolding protein, associates with the tip-link component cadherin 23. J Neurosci 2008; 28(44): 11269–11276

[26] Yonezawa S, Hanai A, Mutoh N, Moriyama A, Kageyama T. Redox-dependent structural ambivalence of the cytoplasmic domain in the inner ear-specific cadherin 23 isoform. Biochem Biophys Res Commun 2008; 366(1): 92–97

[27] Xu Z, Oshima K, Heller S. PIST regulates the intracellular trafficking and plasma membrane expression of cadherin 23. BMC Cell Biol 2010; 11(1): 80

[28] Ule J, Ule A, Spencer J, Williams A, Hu JS, Cline M, Wang H, Clark T, Fraser C, Ruggiu M, Zeeberg BR, Kane D, Weinstein JN, Blume J, Darnell RB. Nova regulates brain-specific splicing to shape the synapse. Nat Genet 2005; 37(8): 844–852

[29] Warzecha CC, Sato TK, Nabet B, Hogenesch JB, Carstens RP. ESRP1 and ESRP2 are epithelial cell-type-specific regulators of FGFR2 splicing. Mol Cell 2009; 33(5): 591–601

[30] Warzecha CC, Shen S, Xing Y, Carstens RP. The epithelial splicing factors ESRP1 and ESRP2 positively and negatively regulate diverse types of alternative splicing events. RNA Biol 2009; 6(5): 546–562

[31] Warzecha CC, Jiang P, Amirikian K, Dittmar KA, Lu H, Shen S, Guo W, Xing Y, Carstens RP. An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition. EMBO J 2010; 29(19): 3286–3300

[32] Keppetipola N, Sharma S, Li Q, Black DL. Neuronal regulation of pre-mRNA splicing by polypyrimidine tract binding proteins, PTBP1 and PTBP2. Crit Rev Biochem Mol Biol 2012; 47(4): 360–378

[33] Romanelli MG, Diani E, Lievens PM. New insights into functional roles of the polypyrimidine tract-binding protein. Int J Mol Sci 2013; 14(11): 22906–22932

[34] Miranda-Rottmann S, Kozlov AS, Hudspeth AJ. Highly specific alternative splicing of transcripts encoding BK channels in the chicken’s cochlea is a minor determinant of the tonotopic gradient. Mol Cell Biol 2010; 30(14): 3646–3660

[35] Mann ZF, Kelley MW. Development of tonotopy in the auditory periphery. Hear Res 2011; 276(1-2): 2–15

[36] Crawford AC, Fettiplace R. An electrical tuning mechanism in turtle cochlear hair cells. J Physiol 1981; 312(1): 377–412

[37] Art JJ, Fettiplace R. Variation of membrane properties in hair cells isolated from the turtle cochlea. J Physiol 1987; 385(1): 207–242

[38] Fuchs PA, Nagai T, Evans MG. Electrical tuning in hair cells isolated from the chick cochlea. J Neurosci 1988; 8(7): 2460–2467

[39] Navaratnam DS, Bell TJ, Tu TD, Cohen EL, Oberholtzer JC. Differential distribution of Ca²⁺-activated K⁺ channel splice variants among hair cells along the tonotopic axis of the chick cochlea. Neuron 1997; 19(5): 1077–1085

[40] Rosenblatt KP, Sun ZP, Heller S, Hudspeth AJ. Distribution of Ca²⁺-activated K⁺ channel isoforms along the tonotopic gradient of the chicken’s cochlea. Neuron 1997; 19(5): 1061–1075

[41] Ramanathan K, Michael TH, Jiang GJ, Hiel H, Fuchs PA. A molecular mechanism for electrical tuning of cochlear hair cells. Science 1999; 283(5399): 215–217

[42] Jones EM, Gray-Keller M, Art JJ, Fettiplace R. The functional role of alternative splicing of Ca²⁺-activated K⁺ channels in auditory hair cells. Ann N Y Acad Sci 1999; 868(1): 379–385

[43] Jones EM, Gray-Keller M, Fettiplace R. The role of Ca²⁺-activated K⁺ channel spliced variants in the tonotopic organization of the turtle cochlea. J Physiol 1999; 518(Pt 3): 653–665

[44] Sakai Y, Harvey M, Sokolowski B. Identification and quantification of full-length BK channel variants in the developing mouse cochlea. J Neurosci Res 2011; 89(11): 1747–1760

[45] Rüttiger L, Sausbier M, Zimmermann U, Winter H, Braig C, Engel J, Knirsch M, Arntz C, Langer P, Hirt B, Müller M, Köpschall I, Pfister M, Münkner S, Rohbock K, Pfaff I, Rüsch A, Ruth P, Knipper M. Deletion of the Ca²⁺-activated potassium (BK) α-subunit but not the BKbeta1-subunit leads to progressive hearing loss. Proc Natl Acad Sci USA 2004; 101(35): 12922–12927

[46] Kubisch C, Schroeder BC, Friedrich T, Lütjohann B, El-Amraoui A, Marlin S, Petit C, Jentsch TJ. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell 1999; 96(3): 437–446

[47] Coucke PJ, Van Hauwe P, Kelley PM, Kunst H, Schatteman I, Van Velzen D, Meyers J, Ensink RJ, Verstreken M, Declau F, Marres H, Kastury K, Bhasin S, McGuirt WT, Smith RJ, Cremers CW, Van de Heyning P, Willems PJ, Smith SD, Van Camp G. Mutations in the KCNQ4 gene are responsible for autosomal dominant deafness in four DFNA2 families. Hum Mol Genet 1999; 8(7): 1321–1328

[48] Kharkovets T, Dedek K, Maier H, Schweizer M, Khimich D, Nouvian R, Vardanyan V, Leuwer R, Moser T, Jentsch TJ. Mice with altered KCNQ4 K⁺ channels implicate sensory outer hair cells in human progressive deafness. EMBO J 2006; 25(3): 642–652

[49] Beisel KW, Rocha-Sanchez SM, Morris KA, Nie L, Feng F, Kachar B, Yamoah EN, Fritzsch B. Differential expression of KCNQ4 in inner hair cells and sensory neurons is the basis of progressive high-frequency hearing loss. J Neurosci 2005; 25(40): 9285–9293

[50] Scheffer DI, Shen J, Corey DP, Chen ZY. Gene expression by mouse inner ear hair cells during development. J Neurosci 2015; 35(16): 6366–6380

[51] Shen J, Scheffer DI, Kwan KY, Corey DP. SHIELD: an integrative gene expression database for inner ear research. Database (Oxford) 2015, 2015:bav071

[52] Long JC, Caceres JF. The SR protein family of splicing factors: master regulators of gene expression. Biochem J 2009; 417(1): 15–27

[53] Deol MS, Gluecksohn-Waelsch S. The role of inner hair cells in hearing. Nature 1979; 278(5701): 250–252

[54] Deol MS. The inner ear in Bronx waltzer mice. Acta Otolaryngol 1981; 92(3-4): 331–336

[55] Bock GR, Yates GK, Deol MS. Cochlear potentials in the Bronx waltzer mutant mouse. Neurosci Lett 1982; 34(1): 19–25

[56] Horner KC, Lenoir M, Bock GR. Distortion product otoacoustic emissions in hearing-impaired mutant mice. J Acoust Soc Am 1985; 78(5): 1603–1611

[57] Inagaki M, Kon K, Suzuki S, Kobayashi N, Kaga M, Nanba E. Characteristic findings of auditory brainstem response and otoacoustic emission in the Bronx waltzer mouse. Brain Dev 2006; 28(10): 617–624

[58] Whitlon DS, Gabel C, Zhang X. Cochlear inner hair cells exist transiently in the fetal Bronx Waltzer (bv/bv) mouse. J Comp Neurol 1996; 364(3): 515–522

[59] Sobkowicz HM, Inagaki M, August BK, Slapnick SM. Abortive synaptogenesis as a factor in the inner hair cell degeneration in the Bronx Waltzer (bv) mutant mouse. J Neurocytol 1999; 28(1): 17–38

[60] Cheong MA, Steel KP. Early development and degeneration of vestibular hair cells in bronx waltzer mutant mice. Hear Res 2002; 164(1-2): 179–189

[61] Bussoli TJ, Kelly A, Steel KP. Localization of the bronx waltzer (bv) deafness gene to mouse chromosome 5. Mamm Genome 1997; 8(10): 714–717

[62] Calarco JA, Superina S, O’Hanlon D, Gabut M, Raj B, Pan Q, Skalska U, Clarke L, Gelinas D, van der Kooy D, Zhen M, Ciruna B, Blencowe BJ. Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein. Cell 2009; 138(5): 898–910

[63] Moayedi Y, Basch ML, Pacheco NL, Gao SS, Wang R, Harrison W, Xiao N, Oghalai JS, Overbeek PA, Mardon G, Groves AK. The candidate splicing factor Sfswap regulates growth and patterning of inner ear sensory organs. PLoS Genet 2014; 10(1): e1004055

[64] Chou TB, Zachar Z, Bingham PM. Developmental expression of a regulatory gene is programmed at the level of splicing. EMBO J 1987; 6(13): 4095–4104

[65] Zachar Z, Chou TB, Bingham PM. Evidence that a regulatory gene autoregulates splicing of its transcript. EMBO J 1987; 6(13): 4105–4111

[66] Zachar Z, Chou TB, Kramer J, Mims IP, Bingham PM. Analysis of autoregulation at the level of pre-mRNA splicing of the suppressor-of-white-apricot gene in Drosophila. Genetics 1994; 137(1): 139–150

[67] Denhez F, Lafyatis R. Conservation of regulated alternative splicing and identification of functional domains in vertebrate homologs to the Drosophila splicing regulator, suppressor-of-white-apricot. J Biol Chem 1994; 269(23): 16170–16179

[68] Sarkissian M, Winne A, Lafyatis R. The mammalian homolog of suppressor-of-white-apricot regulates alternative mRNA splicing of CD45 exon 4 and fibronectin IIICS. J Biol Chem 1996; 271(49): 31106–31114

[69] Lemaire R, Winne A, Sarkissian M, Lafyatis R. SF2 and SRp55 regulation of CD45 exon 4 skipping during T cell activation. Eur J Immunol 1999; 29(3): 823–837

[70] Kiernan AE, Ahituv N, Fuchs H, Balling R, Avraham KB, Steel KP, Hrabé de Angelis M. The Notch ligand Jagged1 is required for inner ear sensory development. Proc Natl Acad Sci USA 2001; 98(7): 3873–3878

[71] Tsai H, Hardisty RE, Rhodes C, Kiernan AE, Roby P, Tymowska-Lalanne Z, Mburu P, Rastan S, Hunter AJ, Brown SD, Steel KP. The mouse slalom mutant demonstrates a role for Jagged1 in neuroepithelial patterning in the organ of Corti. Hum Mol Genet 2001; 10(5): 507–512

[72] Brooker R, Hozumi K, Lewis J. Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear. Development 2006; 133(7): 1277–1286

[73] Kiernan AE, Xu J, Gridley T. The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet 2006; 2(1): 27–38

[74] Verduci L, Simili M, Rizzo M, Mercatanti A, Evangelista M, Mariani L, Rainaldi G, Pitto L. MicroRNA (miRNA)-mediated interaction between leukemia/lymphoma-related factor (LRF) and alternative splicing factor/splicing factor 2 (ASF/SF2) affects mouse embryonic fibroblast senescence and apoptosis. J Biol Chem 2010; 285(50): 39551–39563

[75] Wu H, Sun S, Tu K, Gao Y, Xie B, Krainer AR, Zhu J. A splicing-independent function of SF2/ASF in microRNA processing. Mol Cell 2010; 38(1): 67–77

[76] Meseguer S, Mudduluru G, Escamilla JM, Allgayer H, Barettino D. MicroRNAs-10a and-10b contribute to retinoic acid-induced differentiation of neuroblastoma cells and target the alternative splicing regulatory factor SFRS1 (SF2/ASF). J Biol Chem 2011; 286(6): 4150–4164

[77] Makeyev EV, Zhang J, Carrasco MA, Maniatis T. The microRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol Cell 2007; 27(3): 435–448

[78] Boutz PL, Chawla G, Stoilov P, Black DL. MicroRNAs regulate the expression of the alternative splicing factor nPTB during muscle development. Genes Dev 2007; 21(1): 71–84

[79] Weston MD, Pierce ML, Rocha-Sanchez S, Beisel KW, Soukup GA. MicroRNA gene expression in the mouse inner ear. Brain Res 2006; 1111(1): 95–104

[80] Friedman LM, Dror AA, Mor E, Tenne T, Toren G, Satoh T, Biesemeier DJ, Shomron N, Fekete DM, Hornstein E, Avraham KB. MicroRNAs are essential for development and function of inner ear hair cells in vertebrates. Proc Natl Acad Sci USA 2009; 106(19): 7915–7920

[81] Lewis MA, Steel KP. MicroRNAs in mouse development and disease. Semin Cell Dev Biol 2010; 21(7): 774–780

[82] Ushakov K, Rudnicki A, Avraham KB. MicroRNAs in sensorineural diseases of the ear. Front Mol Neurosci 2013; 6: 52

[83] Moulton VR, Gillooly AR, Tsokos GC. Ubiquitination regulates expression of the serine/arginine-rich splicing factor 1 (SRSF1) in normal and systemic lupus erythematosus (SLE) T cells. J Biol Chem 2014; 289(7): 4126–4134

[84] Venables JP, Dalgliesh C, Paronetto MP, Skitt L, Thornton JK, Saunders PT, Sette C, Jones KT, Elliott DJ. SIAH1 targets the alternative splicing factor T-STAR for degradation by the proteasome. Hum Mol Genet 2004; 13(14): 1525–1534

[85] Zhang L, Tran NT, Su H, Wang R, Lu Y, Tang H, Aoyagi S, Guo A, Khodadadi-Jamayran A, Zhou D, Qian K, Hricik T, Côté J, Han X, Zhou W, Laha S, Abdel-Wahab O, Levine RL, Raffel G, Liu Y, Chen D, Li H, Townes T, Wang H, Deng H, Zheng YG, Leslie C, Luo M, Zhao X. Cross-talk between PRMT1-mediated methylation and ubiquitylation on RBM15 controls RNA splicing. eLife 2015; 4: e07938<?Pub Caret?>

[86] Henzl MT, O’Neal J, Killick R, Thalmann I, Thalmann R. OCP1, an F-box protein, co-localizes with OCP2/SKP1 in the cochlear epithelial gap junction region. Hear Res 2001; 157(1-2): 100–111

[87] Nelson RF, Glenn KA, Zhang Y, Wen H, Knutson T, Gouvion CM, Robinson BK, Zhou Z, Yang B, Smith RJ, Paulson HL. Selective cochlear degeneration in mice lacking the F-box protein, Fbx2, a glycoprotein-specific ubiquitin ligase subunit. J Neurosci 2007; 27(19): 5163–5171

[88] Narimatsu M, Bose R, Pye M, Zhang L, Miller B, Ching P, Sakuma R, Luga V, Roncari L, Attisano L, Wrana JL. Regulation of planar cell polarity by Smurf ubiquitin ligases. Cell 2009; 137(2): 295–307

[89] Van Campenhout CA, Eitelhuber A, Gloeckner CJ, Giallonardo P, Gegg M, Oller H, Grant SG, Krappmann D, Ueffing M, Lickert H. Dlg3 trafficking and apical tight junction formation is regulated by nedd4 and nedd4-2 e3 ubiquitin ligases. Dev Cell 2011; 21(3): 479–491

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg