Project Grant R21DC022058

ALTERNATIVE SPLICING IN THE MOUSE INNER EAR AT SINGLE CELL RESOLUTION - SENSORINEURAL HEARING LOSS AND VESTIBULAR DYSFUNCTION ARE COMMON SENSORY DISORDERS AFFECTING MILLIONS OF INDIVIDUALS WORLDWIDE1,2. THE PRIMARY CAUSE OF BOTH HEARING LOSS AND PERIPHERAL VESTIBULAR DISORDERS IN HUMANS IS DYSFUNCTION OF THE INNER EAR, WHICH CAN OCCUR AS A RESULT OF NOISE EXPOSURE, AGING, GENETIC DEFECTS, ENVIRONMENTAL EXPOSURE TO PATHOGENS, AND AS A SIDE EFFECT OF OTOTOXIC MEDICATIONS. THERE ARE LIMITED THERAPIES FOR SENSORINEURAL HEARING LOSS AND VESTIBULAR DYSFUNCTION, MAINLY DUE TO THE LACK OF UNDERSTANDING OF SOME OF THE MOLECULAR MECHANISMS THAT GOVERN THE HEARING AND BALANCE ORGANS. THERE REMAIN KEY UNANSWERED QUESTIONS ABOUT THE GENETIC ARCHITECTURE AND MOLECULAR MECHANISMS OF TRANSCRIPTIONAL CONTROL USED DURING MAMMALIAN DEVELOPMENT AND WITHIN THE ADULT INNER EAR. RECENTLY, SINGLE CELL RNA SEQUENCING TECHNOLOGY HAS SHOWN THE REMARKABLE TRANSCRIPTIONAL HETEROGENEITY OF CELL TYPES AND DEVELOPMENTAL DYNAMICS OF THE MOUSE INNER EAR3??. HOWEVER, TRANSCRIPTIONAL DIVERSITY AND CONTROL IS ONLY ONE LEVEL OF GENETIC REGULATION. ONE OF THE KEY UNDERLYING MECHANISMS OF TRANSCRIPTOMIC AND PROTEIN DIVERSITY IS THROUGH ALTERNATIVE SPLICING. CELLS USE ALTERNATIVE SPLICING TO DIVERSIFY THEIR NUMBER OF PROTEINS, CHANGE TRANSLATION EFFICIENCY, CONTROL TRANSCRIPT LOCALIZATION, AND MAKE NON-CODING RNAS?,?. ONLY A LIMITED NUMBER OF GENES THAT UNDERGO ALTERNATIVE SPLICING HAVE BEEN IDENTIFIED IN THE INNER EAR AT CELL TYPE RESOLUTION?. UNDERSTANDING ALTERNATIVE SPLICING OF GENES AT THE SINGLE CELL LEVEL IS CRITICALLY IMPORTANT AS ANY FUTURE THERAPIES TARGETING A SPECIFIC GENE MAY HAVE ON/OFF TARGET EFFECTS IN DIFFERENT SUBSETS OF CELLS. WE HYPOTHESIZE THAT THERE IS CELL TYPE-SPECIFIC DIVERSITY OF ALTERNATIVELY SPLICED MRNA WITHIN THE MAMMALIAN INNER EAR. MOREOVER, REGULATION OF ALTERNATIVE SPLICING EVENTS OCCURS THROUGH SPECIFIC RNA BINDING PROTEINS (RBPS). WE WILL TEST THIS HYPOTHESIS THROUGH TWO AIMS: AIM 1 IS TO CHARACTERIZE THE TRANSCRIPTOME-WIDE ALTERNATIVE SPLICING LANDSCAPE OF THE MOUSE INNER EAR AT SINGLE CELL RESOLUTION. USING NEWLY DEVELOPED COMPUTATIONAL TOOLS, WE WILL IDENTIFY ALTERNATIVELY SPLICED GENES WITHIN BOTH THE DEVELOPING AND ADULT MOUSE INNER EAR FOR THE ENTIRE TRANSCRIPTOME. AIM 2 WILL DETERMINE THE ROLE OF RNA BINDING PROTEINS IN REGULATING ALTERNATIVE SPLICING PROGRAMS IN THE INNER EAR AT SINGLE CELL RESOLUTION. REGULATION OF ALTERNATIVE SPLICING EVENTS IS A KEY POST-TRANSCRIPTIONAL MECHANISM THAT CELLS USE TO DETERMINE WHAT SPLICING PROGRAM MRNA GOES THROUGH AS CARRIED OUT BY RNA BINDING PROTEINS. WE WILL CHARACTERIZE THE EXPRESSION OF RBPS WITHIN THE DEVELOPING MOUSE INNER EAR AND LINK THIS TO ALTERNATIVE SPLICING THROUGH COMPUTATIONAL AND VALIDATION TECHNIQUES TO DEVELOP A SPATIAL AND TEMPORAL MAP OF ALTERNATIVE SPLICING PROGRAMS. IN SUM, WE WILL DEFINE THE HETEROGENEITY OF CELL TYPES BASED ON SINGLE CELL ISOFORM-LEVEL DATA DURING DEVELOPMENT AND IN THE ADULT MAMMALIAN INNER EAR, AND WE WILL IDENTIFY THE KEY PLAYERS IN REGULATING ALTERNATIVE SPLICING. THIS WORK WILL D ALTERNATIVE SPLICING REGULATION DURING DEVELOPMENT AND IN THE ADULT INNER EAR THAT WILL BE NECESSARY FOR FUTURE DEVELOPMENT OF TARGETED INNER EAR THERAPIES.