Mechanisms and roles of RNA interference in Cryptococcus neoformans

RNA干扰是由小RNA介导的基因组调控的基本机制，在真核生物中保守存在。它可以通过转录和转录后调控等多种方式发挥作用，在控制基因表达和基因组稳定性方面发挥重要作用。最近的研究表明，RNAi也可以在宿主-病原体相互作用中发挥重要作用。我们对rnai介导的转录调控的了解大多来自对分裂酵母、裂糖酵母的研究。该酵母具有相对简单的RNAi途径和有限数量的目标位点，这些特征使S. pombe成为RNAi一些核心方面的分子分析的良好模型，但也限制了其在理解RNAi途径中形式和功能的全部多样性方面的实用性。为了解决这个问题，我们已经开始研究另一种酵母的RNAi途径，新型隐球菌。这种担子菌是一种机会性的人类病原体（在免疫功能低下的个体中每年造成约100万例脑膜炎病例），在进化上与更常见的子囊菌（如S. pombe）距离较远。有趣的是，C. neoformans似乎有一个更复杂的RNAi通路，根据我们的初步分析，每个核心组分都有两个，Dicer和Argonaute，每个作用于不同的目标。本项目旨在进一步研究RNAi在隐球菌中的功能。我们将描述RNAi相关蛋白复合物的特征，并确定受RNAi调控的基因表达程序，以及调控机制（例如转录与转录后控制）。我们还将研究RNAi是否参与与隐球菌致病性相关的基因表达变化，并寻找任何跨界RNAi的证据，即隐球菌sirna转移到宿主细胞中并在宿主细胞中发挥作用，反之亦然。该项目将提供广泛的分子生物学技术培训，包括许多组学方法，如小RNA测序、CRAC（蛋白质-RNA交联；鉴定RNAi靶转录物）、蛋白质组学（鉴定相互作用的蛋白质）和ChIP-seq（评估沉默是否与染色质变化有关）。通过定期参加实验室会议、期刊俱乐部和研讨会，培养学生的沟通和表达能力。

RNA interference is a fundamental mechanism of genome regulation mediated by small RNAs and conserved across eukaryotes. It can operate in various ways including transcriptional and post-transcriptional regulation, and plays important roles in controlling gene expression and genome stability. Recent studies suggest that RNAi can also play significant roles in host-pathogen interactions. Much of our understanding of RNAi-mediated transcriptional regulation has come from studies in fission yeast, Schizosaccharomyces pombe. This yeast has a relatively simple RNAi pathway with a limited number of target loci - these features have made S. pombe a good model for molecular analysis of some core aspects of RNAi, but also limit its utility for understanding the full diversity of form and function in RNAi pathways. To address this we have begun to investigate RNAi pathways in another yeast, Cryptococcus neoformans. This basidiomycete is an opportunistic human pathogen (responsible for ~1 million cases of meningitis per year in immunocompromised individuals), and is evolutionarily distant from more commonly studied ascomycetes such as S. pombe. Interestingly, C. neoformans appears to have a more complex RNAi pathway, with two of each of the core components, Dicer and Argonaute, each operating on distinct targets according to our preliminary analyses.The aim of this project is to further investigate RNAi function in Cryptococcus. We will characterise RNAi-related protein complexes, and determine the gene expression programs that are regulated by RNAi, and the mechanisms of regulation (e.g. transcriptional versus post-transcriptional control). We will also look at whether RNAi is involved in changes in gene expression associated with Cryptococcus pathogenicity, and look for any evidence of cross-kingdom RNAi, i.e. Cryptococcus siRNAs being transferred to, and functioning in, host cells, or vice versa. The project will provide training in a broad range of molecular biology techniques including a number of -omics approaches such as small RNA sequencing, CRAC (protein-RNA crosslinking; to identify RNAi target transcripts), proteomics (to identify interacting proteins), and ChIP-seq (to assess whether silencing is associated with changes to chromatin). Communication and presentation skills will be developed through regular participation in lab meetings, journal clubs and seminars.