Cochlear transcript diversity and its role in auditory functions implied by an otoferlin short isoform

Area of Science:

• Genomics
• Auditory Neuroscience
• Molecular Biology

Background:

• Alternative gene splicing generates diverse protein isoforms with distinct functions.
• The full spectrum of gene isoforms in the cochlea, particularly in inner hair cells (IHCs), is largely unknown.
• Understanding these isoforms is critical for deciphering auditory function and dysfunction.

Purpose of the Study:

• To comprehensively identify and characterize alternative gene splicing isoforms in the mammalian cochlea.
• To investigate the functional significance of novel otoferlin isoforms in inner hair cell (IHC) synaptic transmission and auditory phenotypes.
• To expand the understanding of molecular mechanisms underlying hearing.

Main Methods:

• Integrated single-cell short-read and long-read RNA sequencing (scRNA-seq).
• Bioinformatic analysis to identify and annotate novel transcripts.
• Experimental validation using RNA-seq, RT-PCR, Sanger sequencing, and mass spectrometry (MS)-based proteomics.
• Functional analysis of otoferlin isoforms in IHCs through genetic manipulation.

Main Results:

• Discovered 236,012 transcripts in the cochlea, with 126,612 previously unannotated.
• Identified and validated novel spliced isoforms, including a short otoferlin isoform.
• Demonstrated that the short otoferlin isoform maintains normal hearing thresholds but impairs sustained IHC exocytosis.
• Uncovered a previously unrecognized role for otoferlin in endocytic membrane retrieval in IHCs.
• Linked otoferlin isoforms to specific IHC functions and auditory phenotypes.

Conclusions:

• This study significantly expands the known transcript repertoire of the cochlea.
• Novel otoferlin isoforms play critical roles in IHC function, synaptic transmission, and membrane dynamics.
• Isoform-level analysis provides a deeper mechanistic understanding of auditory processes and associated disorders.