Abstract
In mechanosensory hair cells (HCs) of the ear, the transcriptional repressor REST is continuously inactivated by alternative splicing of its pre-mRNA. This mechanism of REST inactivation is crucial for hearing in humans and mice. Rest is one of many pre-mRNAs whose alternative splicing is regulated by the splicing factor SRRM4; Srrm4 loss-of-function mutation in mice (Srrm4 bv/bv ) causes deafness, balance defects, and degeneration of all HC types other than the outer HCs (OHCs). The specific splicing alterations that drive HC degeneration in Srrm4 bv/bv mice are unknown, and the mechanism underlying SRRM4-independent survival of OHCs is undefined. Here, we show that transgenic expression of a dominant-negative REST fragment in Srrm4 bv/bv mice is sufficient for long-term rescue of hearing, balancing, HCs, alternative splicing of Rest, and expression of REST target genes including the Srrm4 paralog Srrm3 We also show that in HCs, SRRM3 regulates many of the same exons as SRRM4; OHCs are unique among HCs in that they transiently down-regulate Rest transcription as they mature to express Srrm3 independently of SRRM4; and simultaneous SRRM4-SRRM3 deficiency causes complete HC loss by preventing inactivation of REST in all HCs. Thus, our data reveal that REST inactivation is the primary and essential role of SRRM4 in the ear, and that OHCs differ from other HCs in the SRRM4-independent expression of the functionally SRRM4-like splicing factor SRRM3.
Highlights
REST is a transcriptional repressor of hundreds of genes that are expressed selectively in mature neurons and hair cells (HCs)
We found that transgenic expression of a dominant-negative fragment of REST (DnREST) in HC of Srrm4bv/bv mice rescued hearing, balancing, inner HCs (IHCs), and vestibular HCs (VHCs), and that it restored the expression of REST target genes and the alternative splicing of many SRRM4-regulated exons, including exon 4 of Rest
The Tg(DnRest) mouse line was outcrossed to the Srrm4bv/bv mouse line, and the effects on hearing in the second filial (F2) generation were evaluated by measuring auditory brainstem responses (ABRs) to broadband sounds on P28 and P120
Summary
REST is a transcriptional repressor of hundreds of genes that are expressed selectively in mature neurons and hair cells (HCs). In neurons REST is down-regulated predominantly by transcriptional silencing, whereas in HCs the down-regulation is caused by splicing of an alternative exon of Rest (i.e., exon 4) into the mature mRNA (Fig 1A) (Ballas et al, 2005; Nakano et al, 2018) This alternative splicing event shifts the translational reading frame in Rest and inactivates the encoded protein by truncating it upstream of a repressor domain and several zinc finger domains that are critical for gene silencing activity (Magin et al, 2002). Genetic evidence from both humans and mice indicates that this exon 4–dependent inactivation of REST is crucial for hearing. Genetic deletion of exon 4 (Rest+/ΔEx4) via homologous recombination results in deafness, balance defects, and perinatal degeneration of all HCs (Nakano et al, 2018)
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