RNA modifications fundamentally alter the fate and function of RNA transcripts in cells. For example, RNA modifications signal proteins associated with vital cellular processes such as mRNA translation, degradation and splicing. Creating modifications to expand RNA functionality in novel ways would bolster our understanding of RNA biology. Unfortunately, our ability to engineer modifications to control RNA in vivo is limited. Chemically or chemoenzymatically synthesized RNA must be transfected into cells. In this proposal, I introduce two new concepts that constitute the first genetically encoded novel RNA modifications. The first concept is self-modifying RNA aptamers that bind cofactors to form novel nucleotide modifications. I have discovered RNA aptamers that bind a S-adenosylmethionine analog, called ProSeAM (propargylic selenium- adenosyl-L-selenomethionine). These RNA aptamers react with ProSeAM to form an alkyne chemical group onto RNA for downstream ?click? chemistry. I propose to use this as a highly efficient method for in-cell labeling of RNA for affinity purification of RNA-bound proteins. In this proposal, I describe a strategy to move this in vitro technology into cells, so that these catalytic RNAs (i.e. ribozymes) can be expressed in cells and become spontaneously modified to form alkyne-modified RNA. This will be applied to the XIST transcript, which is a functionally important lncRNA with many suspect and well documented binding proteins. This method will tag exist with a biotin modification which will allow for the removal of any proteins crosslinked to it. The second concept involves expanding the current set of RNA-modifying enzymes. I am developing a new concept in which protein-modifying enzymes are ?tricked? into perform their modifying reactions on RNA. In my approach, I develop RNAs that mimic the peptide substrate of the biotin ligase BirA. By repurposing BirA for the modification of my RNA target, I will study encoded RNAs in living cells. This method will be applied to an mRNA associated with LARP1, a protein important in mRNA translation. Overall, the technologies described in this proposal will provide new strategies to control targeted RNAs so that we can study the mechanisms associated with their cellular functions.
This application discusses projects to encode new modifications and functionalities in RNA. The projects described in the following application will result in the first ribozymes and aptamers which respectively use cofactors and a posttranslational modifying enzyme to create unnatural ribonucleotide ?tags'. The new RNA tagging technology proposed here will be encoded in target RNAs to modify them in vivo, expanding our knowledge of the reactions and biological mechanisms RNA can facilitate.
