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.
