Expression of Cochlin mRNA Splice Variants in the Inner Ear

Abstract

Proteomic analysis of inner ear proteins revealed unique properties of cochlin, encoded by the COCH gene. We detected 3 cochlin isoforms, p63s, p44s and p40s, in the inner ear tissue and a short 16-kDa isoform, cochlin-tomoprotein (CTP), in the perilymph. The role of the cochlin isoforms has not been elucidated. To improve our understanding of the mechanism of cochlin isoform expression, we investigated rat cochlin mRNA expression in the inner ear and other organs. We performed RNA-ligation-mediated amplification of cDNA ends (RLM-RACE) using RNA isolated from the inner ear and spleen of rats, which are known to express abundant cochlin mRNA. We also examined the expression profile of full-length cochlin mRNA by nested RT-PCR in the cerebrum, cerebellum/brain stem, eye, inner ear, thyroid gland, thymus gland, lung, heart, liver, spleen, adrenal gland, kidney and blood. We verified CTP expression in rat perilymph by Western blot. By RLM-RACE, alternately spliced variants of cochlin mRNA with 3 different lengths were detected (2442, 2008 and 724 bp). The two longer mRNAs encode full-length cochlin with different polyadenylation signals in the 3′-untranslated region, which are expressed both in the ear and spleen. The short variant encodes the limulus factor C, cochlin, late gestation lung protein (LCCL) domain and the N-terminal sequence of the von Willebrand factor A (vWFA1) domain, and this variant was detected only in the ear. All 3 variants have the same transcriptional start site. By RT-PCR, we found that full-length cochlin was expressed in all organs examined, with a splice variant in the heart. By Western blot, we detected short isoforms (11–17 kDa) in the perilymph. Cochlin isoform formation is regulated, at least in part, by alternative splicing at the transcriptional level. The short mRNA was detected only in the inner ear, and this variant may provide a clue to understanding the formation and function of cochlin isoforms.

Cochlin expression in the rat perilymph during postnatal development

“…Cochlin appears to have numerous heterogeneous isoforms that are caused partly by N-glycosylation and proteolytic cleavage, and potentially also by alternative splicing [9,10,13,17]. Previous 2D gel investigation identified a full-length isoform, as well as smaller isoforms with truncations of the N terminus.…”

“…The molecular weight of CTP is different in different species, e.g. rat (11)(12)(13)(14)(15)(16)(17), human (16 kDa), and bovine (18-23 kDa) [13]. The importance of cochlin has been shown in a variety of pathological conditions, such as DFNA9, autoimmune hearing loss, glaucoma, and others.…”

“…In our preliminary investigation, it was possible to microscopically identify the perilymphatic space as early as DAB12, and perilymph by itself could be collected without any other tissue contamination. Human, bovine, and rat perilymph contain two cochlin isoforms, the full-length isoform p63 and the short truncated isoform CTP [10,13]. The origin of these cochlin isoforms has been attributed to a variety of mechanisms, including proteolysis of peptide bonds, chemical modifications such as glycosylation, phosphorylation and/or deamination, and multiple transcripts [9,[17][18][19].…”

“…Since human, bovine, and rat perilymph contains two cochlin isoforms, the full-length isoform p63 and the short truncated isoform CTP, we used the rabbit polyclonal anti-LCCL-C Ab (Figure 1), which recognizes these isoforms [10,13]. This antibody was prepared as described previously [10].…”

Cochlin in autoimmune inner ear disease: Is the search for an inner ear autoantigen over?

“…isoforms of cochlin have been described with molecular weights of 40 kDa, 46 kDa and 60 kDa [3,5,6]. In addition, a smaller 16 kDa isoform called cochlin-tomoprotein has been identified in the perilymph.…”