Preliminary Study Of The Role Of ILDR1 And ILDR2 In Alternative Gene Splicing Of Inner Ear

Background and objectiveHearing loss is the most prevalent sensory impairment that affects millions of people in the world,half of which are caused by genetic factors.Till 2015,141 loci associated with non-syndromic hearing loss(NSHL)have been mapped in human genome,and 85 genes have been identified as deafness genes.With the help of new sequencing technologies such as massively parallel sequencing(MPS)or next generation sequencing(NGS),more deafness genes are expected to be uncovered.Mutation of the Ildr1 gene can cause autosomal recessive hearing impairment DFNB42.Immunoglobulin-like domain containing receptor 1(ILDR1)is a putative type ? transmembrane protein containing an immunoglobulin(Ig)-like extracellular N-terminal domain,transmembrane region and intracellular domain.ILDR1 belongs to an evolutionally conserved protein family,angulin protein family,which also includes ILDR2 and LSR.It has been shown that these three proteins are localized at the cell junction(TJ)and are important for the formation of TJs.Ildr1 knockout mice showed profound hearing loss accompanied with postnatal cochlear hair cell degeneration.In mouse inner ear,Ildr1 mRNA was detected in hair cells as well as supporting cells,and ILDR1 protein was shown to localize at tricellular tight junctions(tTJs).Another tight junction protein,Tricellulin,was also required for the structure and function of tTJs,and mutations of Tricellulin gene caused autosomal recessive hearing impairment DFNB49.ILDR1 recruited Tricellulin to tTJs,whereas DFNB42-associated ILDR1 mutant protein fails to do so.ILDR1 has been shown to mediate fat-stimulated cholecystokinin(CCK)secretion,raising the possibility that ILDR1 could act as a signaling receptor.Nevertheless,the role of ILDR1 other than regulating tTJs largely remains unknown at present.Alternative splicing is important for regulation of protein function and proteomic diversity.During alternative splicing,the exons of pre-mRNAs are spliced together in different arrangements to give rise to distinct mature mRNAs,which ultimately produces structurally and functionally distinct protein variants.Alternative splicing deficits cause various types of disease including hearing loss.Transformer 2 protein homolog alpha(TRA2A),transformer 2 protein homolog beta(TRA2B)and serine/arginine-rich splicing factor 1(SRSF1)belong to SR protein family.TRA2A and TRA2B are the homologous proteins of Drosophila Tra-2(the earliest discovered SR protein)in mammals,and SRSF1 is the first identified mammalian SR protein.All of these proteins have been shown to play an important role in alternative splicing.In this paper,we try to elucidate the function of ILDR1 other than tTJs regulation.Our results suggested that ILDR1 could regulate alternative pre-m.RNA splicing via binding to splicing factors TRA2A,TRA2B and SRSF1.Scientific questions to be solvedIn this paper,we need to solve the scientific problems as the following:1)Could ILDR1 participate other functions in addition to tTJs regulation formation?2)Could ILDR1 regulate alternative pre-mRNA splicing?3)Could alternative gene splicing in mice inner ear be affected by knocking out Ildr1?4)Could other members of angulin protein family regulate alternative pre-mRNA splicing?Research programs and the resultsIn order to solve the above scientific problems,we first performed yeast two-hybrid screening of a chicken cochlear cDNA library using the C-terminal intracellular domain of chicken ILDR1 as bait.Among the positive clones identified,several clones encode for a group of splicing factors,including TRA2A,TRA2B,and SRSF1.These three proteins belong to SR protein family,which share one or two serine/arginine-rich domain(RS domain)as well as RNA recognition motif(RRM domain).They have been shown to play important roles in alternative pre-mRNA splicing.We then performed co-immunoprecipitation(co-IP)experiments to confirm the interaction between ILDR1 and the splicing factors and found that the RS domain is responsible for the interaction with ILDR1 with TRA2A,TRA2B and SRSF1.We also found that when cotransfected together with TRA2A-mCherry,TRA2B-mCherry,or SRSF1-mCherry,ILDR1-GFP translocates from the cytoplasm into the nuclei,further validating this interaction.We then examined the expression of Tra2a,Tra2b,and Srsf1 in mouse tissues by RT-PCR and in situ hybridization and found that they were expressed in both hair cells and supporting cells.A strong expression of Tra2b in the spiral ganglion cells was also observed.The expression of these three genes peaked in the inner ear of P15 to P30 in mice and showed a complementary pattern.TRA2A/TRA2B/SRSF1 was involved in the splicing of many alternative exons,so we performed a series of experiments to examine whether ILDR1 might regulate alternative pre-mRNA splicing by binding these three transcription factors.We found in HEK293T cells transfected in vitro,TRA2B and SRSF1 promoted the inclusion of exon 4 of TUBD1,exon 12 of IQCB1 and exon 2 of Pcdh19 minigene,respectively,whereas ILDR1 antagonized the function of TRA2B/SRSF1.Taken together,our data suggested that ILDR1 regulated SRSF1-and TRA2B-mediated alternative pre-mRNA splicing.Unexpectedly,no difference was observed between wild-type and Ildr1 knockout mice.To further examine whether loss of ILDR1 affected alternative splicing,RNA from PO cochlea of wild-type and Ildr1 knockout mice were subjected to RNA-seq analysis,which did not reveal any significant differences in alternative gene splicing.As mentioned above,ILDR1 belonged to evolutionally conserved angulin protein family and its two homologous proteins(ILDR2 and LSR)might has the potential to compensate for some of the functions of ILDR1.RT-PCR and quantitative real-time PCR showed that expression of Ildr2 was greatly increased in the basilar membrane of Ildrl knockout mice compared with wild-type mice.However,the expression of Lsr was not obviously affected by Ildr1 deficiency.In situ hybridization results also confirmed that Ildr2,but not Lsr,was upregulated in the cochlea in Ildrl knockout mice.Then,we examined whether ILDR2 had a similar effect to ILDR1 in regulating alternative pre-mRNA splicing.First,we examined ILDR2 and LSR could be co-IPed together with TRA2A/TRA2B/SRSF1 by performing co-IP experiments.When mCherry-tagged TRA2A/TRA2B/SRSF1 was present,ILDR2 translocated into the nuclei and colocalized with TRA2A/TRA2B/SRSF1,similar to ILDR1,whereas LSR.still remained in the cytoplasm.Immediately after we overexpressed ILDR1/IILDR2 or LSR at the cellular level,similar to ILDR1,ILDR2 inhibited SRSF1-or TRA2B-mediated alternative splicing,whereas LSR did not.Last,we knockdowned the expression of endogenous ILDRl and/or ILDR2 in cultured cells and examined its effect on alternative splicing.Transiently-transfected siRNA downregulated the expression of ILDR1 or ILDR2 specifically in HEK293T cells without affecting each other.As a result,alternative splicing of TUBD1 and IQCB1 was affected in ILDR1 or ILDR2 knockdowned cells.When the expression of both ILDRI or 1LDR2 was downregulated simultaneously with siRNAs,alternative splicing of TUBD1 and IQCB1 was affected to a greater extent.This result strongly supported the role of ILDR1/2 proteins in splicing regulation.Innovation and significanceIn this paper,ILDR1 as the research object,we have explored the biological function of deafness gene Ildr1 in addition to tTJs regulation.We have the innovative research results and significance of the following two points:1.In order to explore the mechanisms of Ildr1,we performed yeast two-hybrid screening of a chicken cochlear cDNA library and found ILDR1 could bind to TRA2A,TRA2B,and SRSF1.ILDR1 regulates SRSF1-and TRA2B-meidated alternative pre-mRNA splicing.2.We did not observe any difference between wild-type and Ildr1 knockout mice.But the expression of Ildr2 is greatly increased in the basilar membrane of Ildr1 knockout mice compared with wild-type mice.Overexpression and interference tests have shown that Ildr1 has the same effect as Ildr1 in regulating splicing.These results indicate that Ildr2 compensates for some of the functions of ILDR1 at a certain level.