RBM24 Regulates Hair Cell Development By Affecting Alternative Splicing And MRNA Stability In Mice

RBM24 is an RNA-binding protein,which plays an important role in the posttranscriptional regulation of gene expression.It can mediate the stability of mRNA by specifically binding to its 3’-UTR.As a splicing factor,it can also regulate the alternative splicing of many genes,which plays an important role in determining the fate of cells.For a long time,the study of RBM24 has mainly focused on the heart and muscles.Loss of RBM24 can cause cardiac hypoplasia and impaired striated muscle differentiation in mice,which may be due to the loss of RBM24 leading to many abnormal splicing events.Preliminary study of our laboratory and collaborators found that RBM24 is specifically expressed not only in the heart and skeletal muscle,but also in the lens,olfactory epithelium,cochlea and vestibular hair cells.Further study found that lack of RBM24 can cause abnormal eye development in mice by affecting the stability and the poly(A)tail length of lens-expressed mRNAs.We successfully established Rbm24flox/flox mice using CRISPR-Cas9 system.By mating with Atoh1Cre/+ mice,we obtained a mouse model that specifically knocked out Rbm24 in the inner ear(conditional knockout mice,cKO mice).The results of ABR test showed that the cKO mice had severely hearing loss as early as postnatal day 18(P18).In addition,the statistics on the number of turns and swimming experiments showed that the balance ability of the cKO mice was also severely impaired.We futher observed the morphology of hair cells and stereocilia through whole-mount immunostaining and scanning electron microscopy.The results showed that the cochlear hair cell stereocilia of the cKO mice were disorganized at P0,the earlist age examined,while the cochlear hair cells begun to be lost from P7 and almost completely lost at P10.However,the phenotype of vestibular hair cells was not serious:we first observed the elongation of the lower row of stereocilia and the disappearance of the staircase morphology at P7,the continuous development to normal length at P14,and the disorganization of stereocilia was increased at P60;but no significant loss of vestibular hair cells was observed at P60.To explore the molecular mechanism,we analysed the transcriptome of the basilar membrane in the control and cKO mice using RNA sequencing,and then verified some selected sequencing results using both basilar membrane mRNAs and vestibular mRNAs by RT-PCR.We found that the stability and the splicing of many deafness genes was changed,which may be the reasons for abnormal development and physiological dysfunction of cKO mice inner ear.In summary,our study proved that RBM24 plays a vital role in mice hearing and balance,which is also of great significance for the treatment of human congenital deafness and balance dysfunction.However,there are still many questions.Whether RBM24 affect these genes directly or indirectly?What are the relationships between these genes?All these need to be futher investigated.