Expression of cochlin mRNA splice variants in the inner ear.
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.
Sekine
Kuwon
K
Department of Otorhinolaryngology, Nippon Medical School, Tokyo, Japan.
Ikezono
Tetsuo
T
Matsumura
Tomohiro
T
Shindo
Susumu
S
Watanabe
Atsushi
A
Li
Lishu
L
Pawankar
Ruby
R
Nishino
Takeshi
T
Yagi
Toshiaki
T
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D007758:Ear, Inner / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D016326:Extracellular Matrix Proteins; D015870:Gene Expression / Q000502:physiology; D014644:Genetic Variation / Q000235:genetics; D006801:Humans; D007438:Introns / Q000235:genetics; D009206:Myocardium / Q000378:metabolism; D010498:Perilymph / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics*; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D013154:Spleen / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
C110668:COCH protein, human; D016326:Extracellular Matrix Proteins; D020033:Protein Isoforms; D011506:Proteins; D012333:RNA, Messenger