Loss of splicing factor IK impairs normal skeletal muscle development

Abstract

Background IK is a splicing factor that promotes spliceosome activation and contributes to pre-mRNA splicing. Although the molecular mechanism of IK has been previously reported in vitro, the physiological role of IK has not been fully understood in any animal model. Here, we generate an ik knock-out (KO) zebrafish using the CRISPR/Cas9 system to investigate the physiological roles of IK in vivo. Results The ik KO embryos display severe pleiotropic phenotypes, implying an essential role of IK in embryonic development in vertebrates. RNA-seq analysis reveals downregulation of genes involved in skeletal muscle differentiation in ik KO embryos, and there exist genes having improper pre-mRNA splicing among downregulated genes. The ik KO embryos display impaired neuromuscular junction (NMJ) and fast-twitch muscle development. Depletion of ik reduces myod1 expression and upregulates pax7a, preventing normal fast muscle development in a non-cell-autonomous manner. Moreover, when differentiation is induced in IK-depleted C2C12 myoblasts, myoblasts show a reduced ability to form myotubes. However, inhibition of IK does not influence either muscle cell proliferation or apoptosis in zebrafish and C2C12 cells. Conclusion This study provides that the splicing factor IK contributes to normal skeletal muscle development in vivo and myogenic differentiation in vitro.

IK is essentially involved in ciliogenesis as an upstream regulator of oral-facial-digital syndrome ciliopathy gene,ofd1

“…In parallel, we analyzed the phenotypes of previously generated homozygous ik knockout zebrafish mutants using CRISPR/Cas9 system [19]. Consistent with the observation of ik morphants, ik mutants also exhibited similar ciliopathy phenotypes.…”

“…The MO sequence of ik was 5'-GGAGCCAGAGGATTAGAGTACACAT-3', as previously described [19] and was obtained from GeneTools (Corvallis, OR, USA). For knockdown of ik, embryos at onecell stage were injected with 4 ng of MO using a PV820 Pneumatic PicoPump (World Precision Instruments, Sarasota, Florida, USA) and reared in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4, and 0.1% methylene blue) at 28.5 °C.…”

“…We used a previously analyzed RNA-seq dataset of ik mutants to evaluate differentially expressed genes (DEGs) [19]. In brief, genome-wide RNA-seq data of zebrafish embryos at 3 dpf were analyzed by eBiogen (Seoul, Korea).…”

“…In mice, homozygous knockout of ik results in embryonic lethality. Although we have previously demonstrated in a zebrafish model that depletion of ik impairs the development of skeletal muscle [19], further studies on the physiological role of IK in vivo still need be more required.…”

Comparative analysis of myoglobin in Cetaceans and humans reveals novel regulatory elements and evolutionary flexibility

“…In addition, this predicted MYOD binding is not found when the CCAC mut 3 mutation of Bassel-Duby et al [14] is examined (rVISTA). Last, the human CCAC-box binds a nuclear factor of 40 kD [14], the approximate size of MYOD (see, for example, [63]; 34.5 kD in Uniprot [64]); in contrast, SP1 has a molecular weight of approximately 80.7 kD [64,65]. Thus, the human CCAC-box may have functions beyond the binding of SP1 that contribute to its' relative importance, compared to the minke CCAC-box.…”