How Cyclophilins both Regulate and are Regulated by RNA

亲环蛋白是一类蛋白质伴侣分子（协助折叠/展开和其他大分子结构的组装/拆卸的分子）和信号分子，存在于生命的各个领域。在人类中，亲环蛋白在许多信号通路中发挥关键作用，参与HIV等病毒的病毒生命周期，并在炎症和癌症中发挥作用。最近，这一蛋白质家族被鉴定为RNA结合蛋白，随后的研究表明RNA调节这一重要蛋白质家族的活性。这些发现为了解亲环蛋白是如何调控的以及它们如何调控健康和疾病中重要的关键细胞过程提供了一个全新的研究途径。本研究项目的目标是确定这些蛋白质结合的特定RNA分子，结合界面是什么样子，并确定这些相互作用在亲环蛋白生物学中的作用。这项研究计划将对对亲环蛋白和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，这将有利于其他人寻求了解RNA/蛋白质相互作用的全部阵列。该计划还大力促进教学，培训和学习，同时推进科学发现。这些活动通过精心设计和深思熟虑的指导策略以及在本科课堂上的重大教学创新来实现，包括开发积极的学习环境和在入门科学课程中引入基于研究的实验室。越来越明显的是，许多蛋白质是由RNA以不可预见的方式调节的。迄今为止，在所有无偏倚的哺乳动物和酵母体内交联研究中，亲环蛋白样结构域（CLD）一直被发现是一个非规范的rna结合结构域。亲环蛋白是一种关键的细胞调节剂，在许多生物学途径中发挥重要作用。它们作为rna结合蛋白的发现表明，rna在亲环蛋白生物学中的新作用已被其他观察所暗示，但尚未得到充分探索。靶点包括亲环蛋白A （CypA）的CLD，一个被充分研究的仅由该结构域组成的CLD的代表，以及它的酵母同源物Cpr1。选择Cyp33进行研究还因为它同时包含RNA识别基序（RRM）和CLD结构域。负责与CLD相互作用的RNA基序将首先通过体外选择策略进行鉴定。体内结合位点将通过细胞内光交联确定，然后对相关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CLDs的情况下，一项无偏倚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。采用的体外结合和酶促策略将促进一组高度验证的功能分离突变体的发展。这些工具将允许CLD活性与转录组水平直接相关，并揭示RNA生命周期的哪些方面受到CLD作用的影响。总的来说，这一全面的研究计划将有助于阐明亲环蛋白在RNA生物学中直接和间接发挥的新作用。

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.