内源siRNA通路调控蓝氏贾第鞭毛虫分化的分子机制研究

Endogenous siRNA (endo-siRNA) is an important kind of small non-coding RNA identified in eukaryote in recent years. It has been found to play important role in many key biological processes, such as the maintenance of the genome stability, development regulation and stress resistance. Giardia lamblia (Giardia) is an early-diverging eukaryote, and has been used as an important model to study the differentiation and meiosis process of eukaryote. The proposer has demonstrated the existence of endo-siRNA in Giardia using the deep-sequencing, GLDICER knockdown and GLAGO co-immunoprecipitation experiments. We found that the expression of endo-siRNA was upregulated in the differentiation process of Giardia, and the differentiation of Giardia was blocked after the knockdown of GLDICER, indicating that the endo-siRNA pathway was involved in the differentiation regulation of Giardia. This study aim at identifying the differentiation regulating endo-siRNAs and their direct targets, and clarified the molecular mechanisms of how endo-siRNAs regulate the Giardia differentiation. This study will provide clue to the function and evolution studies of eukaryotic endo-siRNAs.

内源siRNA是近年来在真核生物中发现的一种重要小RNA，它参与维持基因组稳定性，发育调控及抗逆反应等多种重要生命活动。目前对于内源的siRNA的功能和调控机制的研究还处于起步阶段。蓝氏贾第鞭毛虫（贾第虫）作为一种最原始的真核生物，是一种重要研究模型。申请人在前期研究中通过贾第虫小RNA的高通量测序数据分析，内源siRNA加工相关蛋白GLDICER的沉默实验和内源siRNA结合蛋白GLAGO的免疫共沉淀实验证实了贾第虫中存在内源siRNA。我们发现内源siRNA在贾第虫分化过程中表达逐渐上调，并且沉默GLDICER阻碍了贾第虫分化，提示贾第虫内源siRNA通路参与贾第虫的分化调控。本项目将在前期研究基础上，结合高通量测序和多种体内和体外实验技术确定调控贾第虫分化的内源siRNA及其直接靶标，阐明内源siRNA通路调控贾第虫分化的分子机制，为真核生物内源siRNA功能和进化研究提供新线索。

贾第虫是一种最原始的病原单细胞真核生物，它的细胞分化能在体外诱导完成，非常便于研究分化过程中细胞的生理生化变化。我们大规模测了贾第虫分化过程中的小RNA转录组和长RNA转录组。通过对贾第虫RNA转录组的系统分析以及后续的实验验证，我们在贾第虫中首次鉴定到了2种内源siRNA和6种tRNA来源小RNA，以及376个新的贾第虫lncRNA。2种内源siRNA分别来自贾第虫端粒区转座子和基因组中的三个小RNA编码区。我们发现所有我们鉴定到的小RNA在贾第虫分化过程中都是上调的，这说明这些小RNA可能参与贾第虫的分化过程的调控。通过对内源siRNA产生所必须的蛋白Dicer的沉默，我们发现贾第虫的分化能力受到削弱，这证明了内源siRNA确实可能参与了贾第虫的分化和表面抗原过程调控，进一步分析确认siRNA可能通过靶向Bestatin等蛋白来影响贾第虫分化。以上成果目前已发表在SCI收录的Proc. Nat. Acad. Sci.和 BBA-Mol. Cell Res.杂志上。由于所取得成果的重要性，我们还受邀在Springer出版社出版的“Non-coding RNA and inter-kingdom communication”一书中与人合作撰写了一章来介绍该项目所取得的研究成果。