(326a) Modular Assembly of Designer Puf Proteins for Specific Post-Transcriptional Regulation of Endogenous RNA
Due to their modular structure, RNA-binding proteins (RBP) are an attractive platform for the development of tools needed for the study of eukaryotic transcriptomes as well as the manipulation of RNA for therapeutic purposes. In this work, we utilize the Pumilio and FBF (PUF) homology domain as a scaffold for engineering RNA substrate specificity and functionalize it with a repressor domain for post-transcriptional down-regulation of endogenous mRNA.
PUF proteins are eukaryotic RBPs that are typically involved in translational regulation of developmental genes. The RNA-binding domain of PUF proteins ususlly contains 8 structural repeats, each containing 36 amino acids, two of which contribute to the specificity of the repeat to a corresponding nucleotide in the target RNA sequence. With the elucidation of the “code” for RNA recognition by PUF proteins, it was shown that tailored PUF proteins with predicted specificity could be engineered. In the past several years, engineered PUF proteins with designed specificities were successfully implemented for applications like enhancing or repressing translation, modulating splicing, and imaging of endogenous RNA.
One of the limitations to engineering PUF domains with novel specificities is the lack of a cloning platform capable of rapid and efficient introduction of multiple mutations in separate modules simultaneously. In this study, we report the implementation of “Golden Gate" cloning, a modular, type IIS restriction endonuclease-based cloning approach for engineering PUF-based RBPs. We have created a library in which all 8 structural repeats of human Pumilio 1 homology domain are inserted in separate plasmids, and each of them has 3 more variations for the recognition of a certain nucleotide, according to the published “code”. Thus, our library of PUF repeat modules is potentially capable of generating a PUF protein with a specificity for any RNA sequence of 8nt, given that it can be solubly expressed.
In order to demonstrate the efficiency of this approach, we first used this assembly method for rapid construction of several mutant PUF domains with novel specificities and assayed their binding affinities. By replacing as many as 8/8 repeats using the assembly strategy, we were able to obtain a PUF mutant that binds specifically to its corresponding RNA with a KD in a low nanomolar range. Second, we linked the RNA-binding activity of engineered PUF domains to a repressor activity of tristetraprolin (TTP), a key player in AU-rich element (ARE)-mediated mRNA decay, and used the protein fusion in a functional reporter system to assay the PUF domain activity in HeLa cells. Using a firefly luciferase reporter assay, we observed that TTP-PUF fusion represses gene expression in a sequence-specific and modular manner. Finally, we demonstrated the application of the engineered TTP-PUF fusion proteins in translational knock-down of human vascular endothelial growth factor A protein in HEK293 cell line.
This one-step modular assembly approach for PUF domain engineering would allow greater flexibility and speed at creating PUF proteins with user-defined specificities and thus facilitate the use of PUF-based designer RBPs as a tool in research and therapeutics.