Enhanced Specificity of the Viral Suppressor of RNA Silencing Protein p19 toward Sequestering of Human MicroRNA-122

Abstract

Tombusviruses express a 19 kDa protein (p19) that, as a dimeric protein, suppresses the RNAs silencing pathway during infection by binding short-interfering RNA (siRNA) and preventing their association with the RNA-induced silencing complex (RISC). The p19 protein can bind to both endogenous and synthetic siRNAs with a high degree of size selectivity but with little sequence dependence. It also binds to other endogenous small RNAs such as microRNAs (miRNAs) but with lower affinity than to canonical siRNAs. It has become apparent, however, that miRNAs play a large role in gene regulation; their influence extends to expression and processing that affects virtually all eukaryotic processes. In order to develop new tools to study endogenous small RNAs, proteins that suppress specific miRNAs are required. Herein we describe mutational analysis of the p19 binding surface with the aim of creating p19 mutants with increased affinity for miR-122. By site-directed mutagenesis of a single residue, we describe p19 mutants with a nearly 50-fold increased affinity for miR-122 without altering the affinity for siRNA. Upon further mutational analysis of this site, we postulate that the higher affinity relies on hydrogen-bonding interactions but can be sterically hindered by residues with bulky side chains. Finally, we demonstrate the effectiveness of a mutant p19, p19-T111S, at sequestering miR-122 in human hepatoma cell lines, as compared to wild-type p19. Overall, our results suggest that p19 can be engineered to enhance its affinity toward specific small RNA molecules, particularly noncanonical miRNAs that are distinguishable based on locations of base-pair mismatches. The p19-T111S mutant also represents a new tool for the study of the function of miR-122 in post-transcriptional silencing in the human liver.

A Novel p19 Fusion Protein as a Delivery Agent for Short-interfering RNAs

“…We have previously described the p19-Y73S mutation which completely abrogates siRNA binding. 21 We linked Y73S monomers into a linked dimer construct (2x-Y73S) in the same manner as the 2x-p19 construct and confirmed that the linked Y73S dimer remains incapable of binding siRNA ( Figure 2b ). When the TAT peptide is added to the C-terminus ( Figure 2c ), the fusion construct is able to bind siRNA with low affinity ( K d = 1.1 μmol/l) ( Figure 2e ).…”

“…This dissociation constant is only slightly altered from that of wild-type p19 ( K d = 0.2 nmol/l). 18,21,27 We demonstrate that 2x-p19-TAT is able to deliver siRNA into Huh7 cells through the use of fluorescence microscopy and genetic reporter assays. Imaging the delivery of fluorescently labelled siRNA demonstrates that 2x-p19-TAT facilitates entry of siRNA into the cell ( Figure 3 ).…”

“…21,29,30,31 The utility of p19 has been further enhanced through mutations and protein fusions. 21,32,33,34,35 As reported previously, linking two p19 monomers with a flexible linker gives the protein a higher thermostability and a higher affinity for its bound siRNAs, which are generally beneficial properties for p19's development as a biotechnology tool. 29,32 The linked dimer construct, 2x-p19, gives the opportunity to genetically fuse a single tag or fusion protein to the functional dimer unit, giving unique opportunities for p19-based technology.…”

“…The resulting construct “2x-p19-TAT” was confirmed by sequencing. The nonbinding p19-Y73S control construct was previously described 21 and the 2x-Y373S-TAT construct was cloned using the same primers and restriction enzymes as stated above for the wild-type 2x-p19-TAT construct.…”

Allele-Specific Chemical Genetics: Concept, Strategies, and Applications

“…In an effort to specifically regulate RNA−protein interactions, Pezacki and colleagues engineered a virus protein p19 that is known to bind both siRNAs and miRNAs, albeit in different affinity ( Figure 10C). 133 A T111S mutant of p19 was generated and shown to improve the binding affinity toward miRNAs by 48-fold. The rationale for the improved binding was the bulging (bump) structure of miRNAs that fit better into the mutant p19 pocket ( Figure 10C).…”