Deletion of exons 17 and 18 in prestin’s STAS domain results in loss of function

Satoe Takahashi, Tetsuji Yamashita, Kazuaki Homma, Yingjie Zhou, Jian Zuo, Jing Zheng, Mary Ann Cheatham

Research output: Contribution to journalArticle

Abstract

Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin’s C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin’s exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17–18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17–18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.

Original languageEnglish (US)
Article number6874
JournalScientific Reports
Volume9
Issue number1
DOIs
StatePublished - Dec 1 2019

Fingerprint

Exons
Outer Auditory Hair Cells
Calmodulin
Proteins
Messenger RNA
Cochlea
Binding Sites
Cell Membrane
Calcium
Phenotype

ASJC Scopus subject areas

  • General

Cite this

Takahashi, Satoe ; Yamashita, Tetsuji ; Homma, Kazuaki ; Zhou, Yingjie ; Zuo, Jian ; Zheng, Jing ; Cheatham, Mary Ann. / Deletion of exons 17 and 18 in prestin’s STAS domain results in loss of function. In: Scientific Reports. 2019 ; Vol. 9, No. 1.
@article{01fa332199e244f1b8f7312568d6dc61,
title = "Deletion of exons 17 and 18 in prestin’s STAS domain results in loss of function",
abstract = "Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin’s C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin’s exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17–18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17–18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.",
author = "Satoe Takahashi and Tetsuji Yamashita and Kazuaki Homma and Yingjie Zhou and Jian Zuo and Jing Zheng and Cheatham, {Mary Ann}",
year = "2019",
month = "12",
day = "1",
doi = "10.1038/s41598-019-43343-y",
language = "English (US)",
volume = "9",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Nature Publishing Group",
number = "1",

}

Deletion of exons 17 and 18 in prestin’s STAS domain results in loss of function. / Takahashi, Satoe; Yamashita, Tetsuji; Homma, Kazuaki; Zhou, Yingjie; Zuo, Jian; Zheng, Jing; Cheatham, Mary Ann.

In: Scientific Reports, Vol. 9, No. 1, 6874, 01.12.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Deletion of exons 17 and 18 in prestin’s STAS domain results in loss of function

AU - Takahashi, Satoe

AU - Yamashita, Tetsuji

AU - Homma, Kazuaki

AU - Zhou, Yingjie

AU - Zuo, Jian

AU - Zheng, Jing

AU - Cheatham, Mary Ann

PY - 2019/12/1

Y1 - 2019/12/1

N2 - Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin’s C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin’s exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17–18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17–18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.

AB - Cochlear outer hair cells (OHC) express the motor protein, prestin, which is required for sensitivity and frequency selectivity. Because our previous work showed that a calmodulin binding site (CBS) was located in prestin’s C-terminal, specifically within the intrinsically disordered region, we sought to delete the IDR to study the functional significance of calcium-dependent, calmodulin binding on OHC function. Although the construct lacking the IDR (∆IDR prestin) demonstrated wildtype-like nonlinear capacitance (NLC) in HEK293T cells, the phenotype in ∆IDR prestin knockins (KI) was similar to that in prestin knockouts: thresholds were elevated, NLC was absent and OHCs were missing from basal regions of the cochlea. Although ∆IDR prestin mRNA was measured, no prestin protein was detected. At the mRNA level, both of prestin’s exons 17 and 18 were entirely removed, rather than the smaller region encoding the IDR. Our hybrid exon that contained the targeted deletion (17–18 ∆IDR) failed to splice in vitro and prestin protein lacking exons 17 and 18 aggregated and failed to target the cell membrane. Hence, the absence of prestin protein in ∆IDR KI OHCs may be due to the unexpected splicing of the hybrid 17–18 ∆IDR exon followed by rapid degradation of nonfunctional prestin protein.

UR - http://www.scopus.com/inward/record.url?scp=85065199043&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85065199043&partnerID=8YFLogxK

U2 - 10.1038/s41598-019-43343-y

DO - 10.1038/s41598-019-43343-y

M3 - Article

VL - 9

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 6874

ER -