Identification Of Novel FRMD7 Splice Variants And Functional Analysis Of FRMD7 Transcripts During Neuronal Differentiation

Background and purpose:Idiopathic congenital nystagmus （ICN） is an oculomotor disorder characterized by involuntary horizontal oscillations of the eyes that presents at birth or appears in the first months of life, but does not usually worsen over time. ICN is distinct from other ocular disorders in which nystagmus is acquired later in life （e.g., cataracts, glaucoma, albinism） or is accompanied by eye, brain or other health abnormalities. About 7%-30% of patients have a family history of ICN, the most common pattern of inheritance is X-linked dominant inheritance. Mutations in the human FRMD7 gene （NM₁94277）, which encodes the FERM domain containing protein 7 is associated with X-linked ICN. Approximately 50% of X-linked pedigrees and 5% of sporadic ICN cases have been linked to FRMD7 mutations, and more than 40 FRMD7 mutations have been reported worldwide. Alternative splicing is a widespread phenomenon in eukaryotes. It can produce multiple protein products with variable domain compositions from a single gene. The alternative splicing may change the structure of transcripts and its encoded protein, determines the binding properties, intracellular localization, enzymatic activity, protein stability and posttranslational modifications of a large number of proteins. International genome and transcript sequencing projects have shown that the frequency of alternative splicing seems to relate to organism complexity, and more than 90% of human genes are subject to alternatively spliced. It is noteworthy that alternative splicing is markedly more prevalent in the brain compared to other tissues. Recently, more and more research indicated that alternative splicing is clearly evident in tumors, genetic diseases and many neurological disorders highlight the importance of alternative splicing. The purpose of this study is to identify two novel splice variants of FRMD7 in both humans and mice, and to detect the role of the full length FRMD7 and the two splice variants in process of neuronal development.Methods:The splice variants of FRMD7 were identified by RT-PCR in human NT2 cells and mouse fetal brain. Expression levels and tissue distribution of FRMD7 transcripts in developing human fetal brain were tested by RT-PCR analysis. Relative expression levels in human NTERA-2 （NT2） cell line with all-trans retinoic acid （ATRA） or bone morphogenetic protein-2 （BMP-2） treatment were tested by real-time qPCR. EGFP-tagged recombinant plasmids DNA encoding hFRMD7-FL and DsRed-tagged recombinant plasmids DNA encoding hFRMD7_SV1 were used to co-transfect the human NT2 cells, then immunofluorescence experiments were performed to determine the co-localized of the two fusion proteins. Finally, we co-transfect HA or Myc-tagged recombinant plasmids into NT2 cells, then co-immunoprecipitation analyses were performed to demonstrated that FRMD7-FL and FRMD7-S were interacted with each other. Expression levels of hFRMD7_FL or hFRMD7_SV1 transcript were monitored by real-time qPCR in transfected hFRMD7_SV1 or hFRMD7_FL NT2 cells at the indicated time points. NT2 cells stably transfected with hFRMD7_FL or hFRMD7_SV1, immunofluorescence experiments were performed to determine the co-localized of the two transcripts with classⅢ(3-tubulin, as well as the effects on neurite outgrowth.Results:（1） Cloning of two novel splice variant of the FERM domain containing protein 7 （FRMD7） gene is known to cause major cases of X-linked idiopathic congenital nystagmus （XL-ICN）. （2） hFRMD7_FL and hFRMD7_SV1 transcripts exhibit a similar tissue distribution, with highest levels in the cerebellum; expression of hFRMD7_SV2 is spatial and temporal restriction in cerebellum during brain development. （3） The FRMD7 transcripts were upregulated following ATRA-induced differentiation of NT2 cells. Nevertheless, relative expression levels showed a slight but yet significant tendency to decline in BMP-2 induced NT2 cells. （4） hFRMD7_FL and hFRMD7_SV1 colocalized and interacted with each other. （5） hFRMD7_FL and hFRMD7-SV1 splice variants exhibit strong colocalization with classⅢβ-tubulin in NT2 cells ATRA induced for 5 days. （6） Furthermore, overexpression of hFRMD7_FL in NT2 cells resulted in altered neurite development and upregulation of hFRMD7_SV2.Conclusions:（1） The FRMD7 gene has also been proven to be subject to alternative splicing. The one splice variant named FRMD7_SV1 contains a 45 bp truncation in the fourth exon. Another splice variant named FRMD7_SV2 is the the exons 2,3 and 4 missing and generated a severely truncated protein. （2） Tissue distribution indicate that the functions of FRMD7 splice variants may be related to cerebellum development. （3） ATRA-induced NT2 differentiation experiment revealed that the mRNA expression levels of FRMD7 splice variants increased dramatically, we speculated that the transcripts are involved in the process of neurite outgrowth extension. （4） Our data suggest that hFRMD7_FL and hFRMD7_SV1 form a complex and both transcripts function involved in multiple aspects of neuronal development. （5） hFRMD7_FL and hFRMD7_SV1 splice variants exhibit strong colocalization with classⅢβ-tubulin. indicated that the FRMD7 isoforms carry out functions associated with microtubules and cytoskeleton-associated proteins or play a role in a related molecular pathway. （6） Overexpression of hFRMD7_FL led to the stimulation of hFRMD7_SV1 gene expression in ATRA-untreated NT2 cells, and led to a significant increase in neurite length in differentiating NT2 cells. These findings suggest that the hFRMD7_FL paly an dramatically role in the formation and growth of neurites during neuronal differentiation, and also indicated that the hFRMD7_SV1 isoform may play a supporting role during this process.