Single-Molecule Fluorescence Studies on Mechanisms of Protein Machines Working with Small Regulatory and Viral RNAs

Abstract

Andrew Fire found that a specific gene was shut down with the destruction of messenger RNA (mRNA) when tiny snippets of double-stranded RNA (dsRNA) were injected into Caenorhabditis elegans in 1998. This phenomenon was RNA interference (RNAi), a biological process in the control of global gene expression, and it became one of most momentous biomedical discoveries of the last 30 years. The application of RNAi has revolutionized the studies of eukaryotic gene function in research laboratories, and the therapeutic potential for human disease treatment was also had captured the attention of the biological and medical communities. RNAi-based therapeutics were introduced into the clinical trials just six years after the discovery. Because components of the RNAi machinery are to be regulated extensively, it became a major challenge to understand their molecular mechanisms from the biogenesis to effective pathways. MicroRNAs (miRNAs) are ~22nt nucleotide endogenous RNAs central to RNAi with small interfering RNA (siRNA). RNA transcripts are process by Microprocessors in nucleus to be precursors of miRNAs. We observed the procedure that complexes of recombinant Drosha and DiGeorge syndrome chromosomal region 8 (DGCR8) fused to fluorescence proteins bind on hairpin structured primary miRNA at single-molecule level, and showed that a Microprocessor is composed of one Drosha molecule and two DGCR8 molecules. This stoichiometry issue was remained ambiguous due to the heterogeneous behaviors of Microprocessor components in the previous biochemical studies, but it could be clarified with real-time single-molecular observation. Argonaute proteins are highly conserved central effectors of RNAi among eukaryotes. The processed miRNAs incorporate with Argonaute proteins into the RNA-induced silencing complexes (RISCs), and the RISCs search and act on targets by miRNA sequence complementary. We observed target binding procedures of core-RISCs followed by target cleavage, guide RNA unloading, and stable binding pathways at single-molecule level with recombinant human Argonuate 2 and organic dye labeled RNA substrates. The whole procedure of core-RISC reaction was described with kinetic parameters at each stage including core-RISC recycling. Furthermore, we discovered the stable binding mode of core-RISC and RNA sequence dependency on core-RISC pathway providing a clue for improving siRNA design. Some viruses store their genetic information as long double stranded RNAs (dsRNAs), and they insert the RNA molecules into normal cells for infection. The normal cells should distinguish the exogenous RNAs from their endogenous RNAs and activate an immune system to survive, and one of the strategies is to measure the length of RNA molecules. Because normal cells do not have long dsRNA molecules, viral RNA invasion can be noticed if cells can discriminate the long dsRNA (> 1 kb) molecules. Previous cell studies showed that Melanoma Differentiation-Associated protein 5 (MDA5) would be essential for the process. By observing the formation and disassembling procedure of MDA5 filament on dsRNA in real-time at single-molecule level with an organic dye labeled recombinant MDA5 and dsRNAs, we explained the mechanism how small MDA5s can determine the length of dsRNA more than 100 times longer than them.