布氏锥虫未知CCCH-型锌指蛋白TbZC3H8功能特性的初步分析

Characterization of the hypothetical CCCH-type zinc-finger protein TbZC3H8 in Trypanosoma brucei

目的通过RNAi和蛋白复合物鉴定来初步分析布氏锥虫未知锌指蛋白TbZC3H8的特性及功能。方法运用蛋白数据库和分析软件对TbZC3H8进行序列分析和结构域预测;构建RNAi诱导表达细胞株分析TbZC3H8经RNAi敲低后对锥虫生长的影响,并通过RT-QPCR和Western blot检测RNAi干扰效率;构建异位融合表达myc-TAP标签的TbZC3H8细胞株,采用串联亲和纯化合并质谱鉴定其蛋白复合物组成;采用免疫荧光分析蛋白定位。结果 TbZC3H8的CCCH结构域在动基体原虫中高度保守;RNAi下调TbZC3H8后明显抑制了锥虫复制;TbZC3H8蛋白复合物中包含多种未知蛋白和两种RNA结合蛋白;TbZC3H8蛋白定位于胞质中,血清饥饿和热刺激对其定位无影响。结论 TbZC3H8是锥虫生长必需的,TbC3H8与RNA结合蛋白的相互作用暗示其可能在RNA加工代谢中发挥着作用。

Objective To characterize the properties and functions of putative zinc-finger protein TbZC3H8 in Trypanosoma brucei. Methods Sequence analysis and motifs / domains prediction were performed through the protein database searches. An inducible RNAi cell line was generated to study the effect of TbZC3H8 repression on cell viability , and the RNAi knockdown efficiency was estimated by RT-QPCR and Western blot. The cell line ectopically expressing C-terminal myc-TAP tagged TbZC3H8 was generated and used to identify the composition of TbZC3H8 protein complexes through the combination of tandem affinity purification and mass spectrometric analysis. Subcellular localization of the TbZC3H8 protein was determined by immunofluorescence microscopy. Results The CCCH zinc-finger motif of TbZC3H8 is highly conserved in kinetoplastid parasites. Repression of TbZC3H8 by RNAi resulted in the growth inhibition. The TbZC3H8 protein complex was found to contain both unknown proteins and RNA-binding proteins. TbZC3H8 was found to localize in the cytoplasm and the expression level was not changed upon the heat shock and serum starvation. Conclusion TbZC3H8 is essential for the growth of T.brucei. The binding of ZC3H8 with RNA-binding proteins suggested that ZC3H8 might play a role in RNA processing and metabolism.