How Cyclophilins both Regulate and are Regulated by RNA

亲环素是一类蛋白质伴侣(协助折叠/解折叠和其他大分子结构的组装/拆解的分子)和信号分子家族，存在于生命的各个领域。在人类中，亲环素在许多信号通路中发挥关键作用，参与艾滋病毒等病毒的生命周期，并在炎症和癌症中发挥作用。最近，这个蛋白质家族被鉴定为RNA结合蛋白，随后的研究表明，RNA调节这个重要的蛋白质家族的活性。这些发现提供了一条全新的研究途径，以了解亲环素是如何被调控的，以及它们如何调控对健康和疾病至关重要的关键细胞过程。该研究项目的目标是确定这些蛋白质结合的特定核糖核酸分子，结合界面是什么样子，并确定这些相互作用在亲环素生物学中的作用(S)。这项研究计划将对对亲环素和RNA生物学感兴趣的科学界产生广泛的影响，并导致工具的开发，这些工具将有助于其他寻求了解RNA/蛋白质相互作用的研究。该计划还有力地促进了教学、培训和学习，同时促进了科学发现。这些活动通过精心设计和深思熟虑的指导策略以及本科生课堂上的重大教学创新来进行，包括发展积极的学习环境和在科学入门课程中引入以研究为基础的实验室。亲环素样结构域(CLD)在所有无偏见的哺乳动物和酵母体内均被认为是一个非规范的RNA结合域，迄今已有研究报道。亲环素是一种关键的细胞调节因子，在许多生物途径中发挥着重要作用。它们作为RNA结合蛋白的发现表明了RNA在亲环素生物学中的新作用，其他观察已经暗示了这一点，但到目前为止还没有完全被探索。靶标包括亲环素A的CLD(CypA)，它是CLDs的一个研究很好的代表，只包含这个结构域，以及它的酵母同系物Cpr1。Cyp33也被选为研究对象，因为它同时含有RNA识别基序(RRM)和CLD结构域。负责与CLD相互作用的RNA基序将通过体外选择策略进行初步鉴定。体内的结合部位将使用细胞内光交联法进行鉴定，然后对相关的RNA进行下拉和深度测序。RNA激活CLD酶活性的机制将被阐明。在没有CLD的情况下，无偏见的转录组分析将揭示与CLD相关的转录本是否在体内受到直接影响。所采用的体外结合和酶策略将有助于开发一组高度有效的功能分离突变体。这些工具将允许CLD活动与转录组水平直接相关，并还可以揭示CLD活动影响RNA生命周期的哪些方面。总体而言，这项全面的研究计划将有助于阐明亲环素在RNA生物学中直接和间接扮演的新角色。

Cyclophilins are a family of protein chaperones (molecules that assist in the folding/unfolding and the assembly/disassembly of other macromolecular structures) and signaling molecules found in every domain of life. In humans, cyclophilins play key roles in numerous signaling pathways, are involved in viral life cycles for viruses such as HIV, and play roles in inflammation and cancer. Recently, this family of proteins has been identified as RNA-binding proteins, and subsequent studies suggest RNA regulates the activity of this important family of proteins. These findings provide a wholly new avenue of study to understand how cyclophilins are regulated and how they regulate key cellular processes important in health and disease. The goal of this research project is to identify the specific RNA molecules to which these proteins bind, what that binding interface looks like, and to determine the role(s) of these interactions in cyclophilin biology. This research program will have a broad impact on the scientific communities interested in cyclophilin and RNA biology and lead to the development of tools that will benefit studies by others that seek to understand the full array of RNA/protein interactions. This program also strongly promotes teaching, training and learning while advancing scientific discovery. These activities occur through well-designed and thoughtful mentoring strategies as well as through significant teaching innovations in the undergraduate classroom, including development of active learning environments and introduction of research-based labs in introductory science courses.It is becoming apparent that many proteins are regulated by RNA in unforeseen ways. The cyclophilin-like domain (CLD) is consistently revealed as a non-canonical RNA-binding domain in every unbiased mammalian and yeast in vivo cross-linking study reported to date. Cyclophilins are a key cellular regulator and play important roles in numerous biological pathways. Their discovery as RNA-binding proteins suggests novel roles for RNAs in cyclophilin biology which have been hinted at by other observations, but as yet have not been fully explored. Targets include the CLD of cyclophilin A (CypA), a well-studied representative of CLDs that consists solely of this domain, and its yeast homolog Cpr1. Cyp33 was also selected for study because it contains both an RNA recognition motif (RRM) and CLD domain. RNA motifs responsible for interacting with the CLD will be identified initially by using in vitro selection strategies. In vivo binding sites will be identified using in cell photocrosslinking followed by pull down and deep sequencing of the associated RNAs. The mechanism of RNA activated CLD enzymatic activity will be elucidated. An unbiased transcriptome analysis in the absence of CLDs will reveal whether the transcripts found to be associated with CLD are directly impacted in vivo. The in vitro binding and enzymatic strategies employed will facilitate the development of a set of highly validated separation-of-function mutants. These tools will allow the correlation of CLD activity directly with transcriptome levels and also to uncover what aspects of the RNA life cycle are impacted by CLD action. Overall, this comprehensive research plan will help elucidate the novel roles cyclophilins play both directly and indirectly in RNA biology.