Establishment Of Drosophila In Situ Reporter Gene Of LncRNA CR43356 And Preliminary Study On Its Function Of Regulating Apoptosis

Long non-codingRNA（lncRNA）is a transcript with sizes greater than 200nucleotides in length,and has no obvious protein coding potential.Over the past few decades,we have discovered many lncRNA with different sequences,structures,and functions.Emerging evidence suggests that lncRNA plays a vital role in diverse and dynamic cellular processes and is an important molecule for RNA to achieve function.Therefore,a comprehensive understanding of the function of lncRNA will greatly promote our current understanding of various cellular regulatory networks and disease mechanisms.In recent years,the great development of technology makes it possible to discover a large number of lncRNA.However,it remains difficult to identify and characterize lncRNA due to the globally low expression of lncRNA.The cellular localization and distribution of lncRNA is closely related to its physiological function.Therefore,understanding the cellular localization of a particular lncRNA provides important insights into its biogenesis and function.At present,fluorescence in situ hybridization is an important method to detect the spatial distribution of lncRNA at the subcellular level.In recent years,with the continuous improvement of CRISPR-Cas9 technology,researchers have invented a scarless and accurate editing technology for genome,which not only expands the fly genome research toolkit,but also provides an important tool for further exploring the function of lncRNA.Here,we used two-step cassette exchange to establish in situ reporter gene fly for better understand the function of lncRNA CR43356.First,we replaced the 346bp fragment inside CR43356 with an excisable compact y^wing2+cassette using CRISPR making a null allele.Second,the y^wing2+cassette was removed using CRISPR and replaced with the EGFP cassette by homology directed repair（HDR）.Compared with the traditional method of generating null alleles based on CRISPR-Cas9,this method allows to completely replace the target gene and generate null alleles.Previous experimental data showed that CR43356 participated in the regulation of apoptosis.Here,we established CR43356 null allele and in situ reporter gene Drosophila,which provided an important tool for further study of the function of CR43356,and preliminarily explored the mechanism of apoptosis induced by the deletion of CR43356.The mechanism of apoptosis is highly conservative in the whole biological evolution process,and caspase family proteins are the core of apoptosis regulation.The characteristic pattern of cell apoptosis during the development of Drosophila has been well characterized.Therefore,flies provide an excellent model system for recognizing the conservative mechanisms regulating apoptosis.In the process of apoptosis,the inhibitor of apoptosis protein（IAP）antagonists RHG proteins（Reaper,Hid and Grim）bind to Drosophila inhibitor of apoptosis protein 1（Diap1）,promoting its ubiquitination and degradation,thus releasing the inhibition of the caspases.The removal of Diap1 promotes the activation of the initial caspase,activates the effector caspase,then executes the apoptosis process.By using the deficiency line Df（3L）H99,we found that the cell apoptosis induced by the deletion of CR43356 was significantly reduced,indicating that the RHG gene played an important role in the apoptosis induced by CR43356 deletion.Further studies found that the hid was necessary for apoptosis induced by CR43356deletion,but not grim and rpr.In addition,our observations showed that the expression level of Diap1 protein in CR43356 deletion fly was significantly reduced.Based on these results we demonstrated that CR43356 can inhibit the expression of Hid,which is helpful to maintain the normal level of Diap1 in cells.