黑腹果蝇头部中RNA结合蛋白RBP9的互作蛋白筛选

【目的】筛选RNA结合蛋白RBP9在黑腹果蝇Drosophila melanogaster头部中的互作蛋白。【方法】利用CRISPR/Cas9基因组编辑技术，分别将3×Flag和V5标签编码序列插入到黑腹果蝇成虫头中RBP9和FNE基因起始密码子ATG的后面，构建黑腹果蝇转基因品系3×Flag-RBP 9/3×Flag-RBP9(3FRBP9)和V5-FNE/V5-FNE(VFNE)；利用免疫沉淀和质谱法鉴定黑腹果蝇3FRBP9和野生型品系成虫头部中RBP9的互作蛋白并进行生物信息学分析。利用免疫共沉淀方法检测RBP9与FNE蛋白之间的相互作用。【结果】质谱法从黑腹果蝇成虫头部共鉴定了190个与RBP9相互作用的蛋白，其中包括ELAV和FNE。KEGG富集分析显示这些蛋白的基因主要参与核糖体、碳代谢、三羧酸循环、氨基酸生物合成等通路。免疫共沉淀实验结果验证了RBP9与FNE蛋白之间存在相互作用。【结论】RNA结合蛋白ELAV家族成员在黑腹果蝇成虫头部中具有相互作用。本研究为RBP9在果蝇神经系统发育中的功能研究提供了重要实验依据。

Screening of the interacting proteins of RNA-binding protein RBP9 in the head ofDrosophila melanogaster

【Aim】 To screen the interacting proteins of RNA-binding protein RBP9 in the head of Drosophila melanogaster. 【Methods】 By using the CRISPR/Cas9 genome editing technology, the coding sequences of 3×Flag and V5 tag were inserted after the start codon ATG of RBP9 and FNE in the head of D. melanogaster adults, respectively, and 3×Flag-RBP9/3×Flag-RBP9 (3FRBP9) and V5-FNE/V5-FNE (VFNE) transgenic D. melanogaster strains were constructed. The interacting proteins of RBP9 in the head of adults of 3FRBP9 and the wild-type strain of D. melanogaster were identified by using immunoprecipitation and mass spectrometry and subjected to bioinformatics analysis. The interaction between RBP9 and FNE proteins was analyzed by immune coprecipitation method. 【Results】 A total of 190 proteins interacting with RBP9, including ELAV and FNE, were identified in the head of D. melanogaster adults by mass spectrometry. KEGG enrichment analysis showed that the genes of these proteins are mainly involved in ribosome, carbon metabolism, tricarboxylic acid cycle, biosynthesis of amino acids and other pathways. The results of immune coprecipitation test validated the interaction between RBP9 and FNE proteins. 【Conclusion】 RNA-binding proteins of the ELAV family interact with each other in the head of D. melanogaster adults. This study provides an important experimental basis for the further functional study of RBP9 in the nervous system development of Drosophila.