A Mechanosensitive Channel, Mouse Transmembrane Channel-Like Protein 1 (mTMC1) Is Translated from a Splice Variant mTmc1ex1 but Not from the Other Variant mTmc1ex2

Abstract

Mechanical stimuli caused by sound waves are detected by hair cells in the cochlea through the opening of mechanoelectrical transduction (MET) channels. Transmembrane channel-like protein 1 (TMC1) has been revealed to be the pore-forming component of the MET channel. The two splice variants for mouse Tmc1 (mTmc1ex1 and mTmc1ex2) were reported to be expressed in the cochlea of infant mice, though only the sequence of mTmc1ex2 had been deposited in GenBank. However, due to the presence of an upstream open reading frame (uORF) and the absence of a typical Kozak sequence in mTmc1ex2, we questioned whether mTMC1 was translated from mTmc1ex2. Therefore, in this study, we evaluated which splice variant was protein-coding mRNA. Firstly, the results of RT-PCR and cDNA cloning of mTmc1 using mRNA isolated from the cochlea of five-week-old mice suggested that more Tmc1ex1 were expressed than mTmc1ex2. Secondly, mTMC1 was translated from mTmc1ex1 but not from mTmc1ex2 in a heterologous expression system. Finally, analyses using site-directed mutagenesis revealed that the uORF and the weak Kozak sequence in mTmc1ex2 prevented the translation of mTMC1 from mTmc1ex2. These results suggest that mTmc1ex1 plays a main role in the expression of mTMC1 in the mouse cochlea, and therefore, mTmc1ex1 should be the mRNA for mTMC1 hereafter.

The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells

“…As we previously reported [23], and others also confirmed using hTMC1 [28], among the two major splice variants for mTmc1 mRNA, mTmc1ex1 is the splice variant which can translate the protein of mTMC1. Therefore, we used mTmc1ex1 as the cDNA of mTmc1 in this study.…”

“…We used mTmc1ex1 cDNA which was cloned from cochleas of male C57BL/6N mice in pcDNA3.1(+) vector (Takara Bio, Otsu, Japan), mammalian expression vectors, as reported previously [23]. Mouse Tmc2 cDNA was amplified from a cDNA library of dorsal root ganglia of a C57BL/6J male mouse and cloned into pEGFPC1 vector (Takara Bio).…”

“…The supernatants were mixed with equal volume of urea sample buffer (8 M urea, 5% SDS, 0.1% bromophenol blue, 0.2 M Tris�Cl (pH = 6.8), and 0.1 M DTT (dithiothreitol) [24]) and heated at 37˚C for 10 min. This treatment dissociated mTMC1 protein aggregates and increased the detection of monomeric mTMC1 proteins as described previously [23]. Proteins (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane.…”

Alternative Splicing of Three Genes Encoding Mechanotransduction-Complex Proteins in Auditory Hair Cells

“…First, we found that Tmc1 starts at exon 1 (and then skips exon 2) or alternatively starts at exon 2 (data not shown) as previously reported ( Kawashima et al, 2011 ), the two isoforms are designated as TMC1A (starting at exon 1) and TMC1B (starting at exon 2). TMC1A is considerably more abundant than TMC1B (ratio ∼95:5) and is therefore considered to be the canonical form ( Yamaguchi et al, 2020 ). More importantly, we identified two previously undescribed alternative splicing events: exon 9 skipping and alternative 3′ splicing in exon 14 at chr19:20 823 987 ( Figs.…”

“…The two isoforms of TMC1 featuring alternative translation initiation sites (TMC1A and TMC1B) differ only within the first 5 amino acid residues at the N terminus ( Fig. 4 ), and although the functional difference between the two isoforms remains unclear, it is expected to be minor ( Yamaguchi et al, 2020 ). Both isoforms can rescue the transducer current in TMC1-knock-out mice ( Kawashima et al, 2011 ; Nist-Lund et al, 2019 ; Cunningham et al, 2020 ).…”