Engineering Chimeric RNA-Binding Proteins: Modular Design, Novel Function

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 our current work, we utilized the Pumilio and FBF (PUF) homology domain as a scaffold for engineering RNA specificity and functionalized it separately with two functional domains: a repressor domain for translational gene expression control as well as with a “motor” domain for intracellular mRNA transport. 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. We first implemented the “Golden Gate" cloning for engineering sequence-specificity of PUF-based RBPs.  Next, by linking the RNA-binding activity of engineered PUF domains to a repressor activity of tristetraprolin (TTP), we sequence-specifically knocked-down endogenous human vascular endothelial growth factor A protein by 77% in the HEK293 cell line. In a following work, we created a biosynthetic device for intracellular RNA transport and local protein translation. We observed a ligand-dependent transport of both RNA-binding protein and target mRNA only in the presence of binding sites in the 3’ UTR of mRNA in HeLa cells. We envision future implementation of these and other PUF-based chimeric RBPs in research and therapeutics.