CCAR-1 Affects Hemidesmosome Biogenesis By Regulating Unc-52/perlecan Alternative Splicing

C.elegans hemidesmosome(CeHD)is a trans-epidermal attachment structure that connects muscles to the cuticle.Hemidesmosomes were previously regarded as stable cell attachment complexes.However,it is increasingly evident that hemidesmosomes are dynamic structures with delicately regulated assembly process and signaling potentials and play important roles in diverse processes,such as mechanotransduction and innate immune response.The detailed process of hemidesmosome assembly and the mechanism regulating hemidesmosome dynamics still remain unclear.The goal of our studies is to document in detail the CeHD biogenesis process during embryonic and larval development,and to elucidate the molecular mechanism regulating CeHD biogenesis.The strategies that we used for this research are as followed: 1)We firstly recorded the reorganization process of CeHDs by GFP fusion protein labeling.2)From a genome-wide RNAi screen searching for factors affecting CeHDs maturation,we identified the CeHD phenotype of a candidate gene ccar-1 mutant by measuring the embryonic lethality and immunostaining of Ce HDs.3)We analyzed the homology between CCAR-1 and mammalian CCAR1 through alignment and examined the expression pattern of CCAR-1 by constructing GFP fusion transgenic strains.4)We utilized genetic interaction,microarray,QPCR,and immunostaining techniques to research the downstream mechanism of how CCAR-1 affects the CeHD biogenesis.5)We furtherly explored the mechanism how CCAR-1 affects unc-52 mRNA alternative splicing by IP-MS,coIP,co-localization and RNAi experiments.The results we obtained are summarized below: 1)We found that embryonic CeHDs underwent dynamic reorganization process during the embryonic development,initially forming puncta along the anterior-posterior axis,then progressively aligning into parallel lines oriented along the circumference,finally fusing together to form continuous stripes.Moreover,at the late embryo stage,CeHD stripes and actin bundles switched from alternating position into overlapping position.And at the L3 larva stage,one CeHD stripe between two furrows was devided into two.2)Through the genomic RNAi screen,we identified a transcription factor CCAR-1/CCAR1 as a potential regulator of CeHD biogenesis.Compare to single mutations,the ccar-1(gk433)mutation combined with the weak viable mutation vab-10A(e698)greatly increased lethality occurring before or soon after hatching and resulted in CeHD defects and muscle detachment.3)Sequence alignment revealed that CCAR-1 is highly homozygous to human CCAR1.We constructed translational GFP fusions of CCAR-1 isoforms and found CCAR-1 isoforms were localized in the nuclei of most cells with different sub-nuclear pattern.4)By genetic interaction analysis of CCAR-1 and CeHD-related components,we identified UNC-52/perlecan as the molecular target of CCAR-1 in CeHDs.Furthermore,we found that loss of CCAR-1 inhibited the alternative splicing of unc-52 exon 17 and thereby increased the unc-52 isoform containing exon 17.Accumulation of exon 17-containing isoform of UNC-52 caused by mec-8 RNAi also resulted in CeHD disruption in vab-10A(e698)background,while knockdown of alternative splicing factor smu-2 expression to decrease the exon 17-containing isoform of UNC-52 partially rescued the defects of ccar-1(gk433);vab-10A(e698)double mutants.These data suggest that the accumulation of unc-52 isoform containing exon 17 indeed damaged CeHD integrity in embryos.The localization analysis of UNC-52 isoforms with or without exon 17-18 suggested that the splicing of unc-52 exon 17-18 may change the binding property of UNC-52 and thereby affect the Ce HD biogenesis.5)We identified HRP-2,the C.elegans homolog for hnRNPQ/R as CCAR-1 binding partner through IP-MS analysis.HRP-2 was previously reported to inhibit the alternative splicing of unc-52 exon17 to generate more exon 17-containing isoforms.We found that HRP-2 and CCAR-1A co-localized in the sub-nuclear compartments,and that HRP-2 physically interacted with CCAR-1 in co-IP experiments.Sequentially,we found inactivation of hrp-2 by RNAi significantly rescued the embryonic lethality of vab-10A(e698);ccar-1(gk433),suggesting that CCAR-1 might suppress the function of HRP-2 in regulating unc-52 splicing by directly binding to HRP-2 protein.Taken together,we propose that CCAR-1 affects the biogenesis of CeHDs by directly regulating the alternative splicing of mRNA encoding ECM protein UNC-52/perlecan.So far,our knowledge about mammalian CCAR1 is limited to the function of CCAR1 as a transcriptional co-activator in cell cycle,division and apoptosis.The function of CCAR1 in affecting cellular attachment structures and mRNA alternative splicing is relatively unappreciated.Therefore,our findings should help to reveal the functions and mechanisms of how CCAR1 regulates mRNA splicing and the integrity of attachment structures in the mammalian systems.