PfRNase II介导恶性疟原虫变异基因转录后调控的机制研究

Malaria is one of the three most severe infectious diseases in the world currently, and Plasmodium falciparum accounts for approximately one million death annually. The key virulence genes associated with immune evasion and pathogenesis of malaria parasites are those variant gene families such as var and rif genes with mutually exclusive expression mode. It has been showed that such expression mode involves epigenetic pathways including chromatin modification at the transcriptional level. Recently, we have reported a novel post-transcriptional regulation of var gene family mediated by PfRNase II, but the underlying mechanism is still not known yet. In this study, to discover the co-regulation network of all of the variant gene families, we aim to investigate the molecular mechanism of the PfRNase II-mediated post-transcriptional regulation pathway by techniques including efficient gene edit, chromatin immunoprecipitation, identification of protein-binding RNAs, and deep sequencing, then integrate the finding with previous known mechanisms of the chromatin modification factors at transcriptional level. We believe that this study will contribute to the elucidation of the mechanisms controlling antigenic variation, immune evasion, and pathogenesis by providing more experimental evidences, and also help to the development of novel intervention tools against falciparum malaria by providing new targets and strategies.

疟疾是当今严重威胁人类健康的三大传染病之一，每年近百万疟疾患者的死亡主要是由恶性疟原虫感染引起，而具有排他性表达模式的var、rif等变异基因家族是其免疫逃避和致病相关的关键基因。研究表明，这种表达模式主要是由表观遗传学因素如染色质修饰等在基因转录水平上进行调控。最近，我们首次报道了恶性疟原虫var变异基因家族存在由PfRNase II分子介导的转录后调控途径，但是具体的分子机制尚不清楚。本课题拟利用高效基因编辑技术、染色质免疫共沉淀、蛋白质结合RNA的分离及核酸深度测序等技术，对PfRNase II在转录后水平上调控恶性疟原虫各类变异基因家族表达和变异的的分子机制进行系统而深入的探索研究，并结合已知染色质修饰因子在转录水平上的调控作用，揭示此类基因家族在不同层面上的协同调控作用，为最终阐明恶性疟原虫免疫逃避和致病机制提供新的实验和理论依据，也将为恶性疟疾的防治提供新的靶点和思路。

疟疾是当今严重威胁人类健康的三大传染病之一，恶性疟原虫感染导致的恶性疟疾是导致每年近百万疟疾患者的死亡的主要原因，而具有排他性表达模式的var、rif等变异基因家族是其免疫逃避和致病相关的关键基因。研究表明，这种表达模式主要是由表观遗传学因素如染色质修饰等在基因转录水平上进行调控。我们在前期工作中首次报道了恶性疟原虫var变异基因家族存在由RNA降解酶PfRNase II介导的转录后调控途径，但是具体的分子机制尚不清楚。本课题拟利用高效基因编辑技术、染色质免疫共沉淀、蛋白质结合RNA的分离及核酸深度测序等技术，对恶性疟原虫各类变异基因家族表达的转录后调控机制进行深入研究。本研究表明：（1）在恶性疟原虫核算外切酶exosome复合物的关键催化亚基中，PfRNase II主要负责重症疟疾相关的upsA亚类var基因的转录后调控；全面负责各类变异基因转录调控的是Rrp6亚基；（2）TRIBE-seq和RIP-seq结果表明，Rrp6调控各类变异基因表达的主要机制是通过实时降解非编码RNA（RUF6）的新生RNA，维持RUF6的排他性表达而实现的；（3）当Rrp6表达水平下降时，RUF6的新生转录产物得以累积，触发局部的染色体变构，从H3K9me3修饰转向H3K9ac修饰（常染色质化），并进而引起其基因一侧的变异基因启动子的活化；（4）过表达的RUF6还能通过核内染色体的空间互作，激活染色体远端的变异基因。因此，疟原虫外切酶复合物中的RNase II与Rrp6协同作用，从不同层面共同控制各类变异基因家族的排他性表达。本研究首次从基因转录后表达调控角度，阐明了恶性疟原虫变异和致病基因家族的排他性表达调控机制，为最终阐明恶性疟原虫极其复杂的免疫逃避和致病机制提供新的实验和理论依据，也将为恶性疟疾的防治提供新的靶点和思路。