Developing the P19 Protein as a Tool for Studying the RNA Silencing Pathway

Abstract

RNA silencing is a cellular mechanism of post-transcriptional gene regulation which is highly conserved among the plant and animal kingdoms of life, and plays a critical part of developmental biology, maintenance of homeostasis, and host-pathogen interactions. The pathway is engaged by small double-stranded (ds)RNA molecules (small RNAs), which effect sequence specific gene silencing by targeting complementary RNA sequences. There are several classes of small RNAs which engage the pathway. MicroRNAs (miRNAs) are expressed in the genome as endogenous regulators of gene expression. Short-interfering RNAs (siRNAs) are usually from exogenous sources such as viral-derived short-interfering RNAs, or synthetic siRNAs which are applied to cells or organisms to inhibit expression of specific genes. The p19 protein is a viral suppressor of RNA silencing (VSRS) endogenous to tombusviruses, which binds small RNA duplexes of any sequence with extremely high affinity. Because of its unique binding properties, recombinant p19 proteins are an excellent platform for tool development surrounding the RNA silencing pathway and are used extensively in novel applications for modulating the activity of small RNAs in living systems and for detecting small RNAs in biological samples. Herein we present work that has increased the breadth of p19’s utility as a biotechnology tool in three distinct realms. First, we present a chemical biology approach which combines p19 and small molecules for potent inhibition of the RNA silencing pathway in human cells. Secondly, we present the development of a novel fusion protein between p19 and a cell penetrating peptide (CPP), which functions as an siRNA delivery agent to allow gene knockdown in human cells. Thirdly, we have improved the utility of p19 for detecting and sequestering human miRNAs through rationally designing the binding surface; we describe mutations which dramatically enhance p19's affinity for human miRNA-122. The work presented here adds to the growing repertoire of engineered RNA binding proteins (RBPs) as tools for studying small RNA molecules and modulating their activity for applications in human therapeutics.