A Dynamic Search Process Underlies MicroRNA Targeting

Abstract

Summary Argonaute proteins play a central role in mediating post-transcriptional gene regulation by microRNAs (miRNAs). Argonautes use the nucleotide sequences in miRNAs as guides for identifying target messenger RNAs for repression. Here we used single-molecule FRET to directly visualize how human Argonaute-2 (Ago2) searches for and identifies target sites in RNAs complementary to its miRNA guide. Our results suggest that Ago2 initially scans for target sites with complementarity to nucleotides 2–4 of the miRNA. This initial transient interaction propagates into a stable association when target complementarity extends to nucleotides 2–8. This stepwise recognition process is coupled to lateral diffusion of Ago2 along the target RNA, which promotes target search by enhancing the retention of Ago2 on the RNA. The combined results reveal the mechanisms that Argonaute likely uses to efficiently identify miRNA target sites within the vast and dynamic agglomeration of RNA molecules in the living cell.

Manipulating MicroRNAs in Murine Models: Targeting the Multi-Targeting in Epilepsy

“…which goes through a nuclear RNase-mediated processing event mediated by the Drosha microprocessor followed by cytoplasmic cleavage of the pre-microRNA by Dicer, resulting in the mature, doublestranded microRNA (~22 nucleotides) (10,11). One strand of the mature microRNA is selected by the protein Argonaute-2, forming the so-called RNA-induced silencing complex (RISC), which then begins to search mRNAs for a region of sufficient complementarity upon which stable base-pair binding occurs (12). It requires only a 7 to 8 nucleotide "seed" region match for microRNA to mRNA targeting effects to occur; this is why microRNAs have so many potential targets and is the source of their multi-targeting property.…”

Approaching the Golden Fleece a Molecule at a Time: Biophysical Insights into Argonaute-Instructed Nucleic Acid Interactions

“…Single-molecule studies now reveal that AGO proteins accelerate target RNA binding by up to 250-fold, when compared to the kinetics of protein-free nucleic acids (Salomon et al, 2015;Chandradoss et al, 2015;Jo et al, 2015). Target binding is initiated by interactions involving as little as three nucleotides within nt 2 to 5 of the guide, with increasing importance toward the 5 0 end of the guide (Salomon et al, 2015;Chandradoss et al, 2015). This indicates that the actual ''seed'' for target binding is smaller than previously annotated.…”

“…The arrangement of the seed in an A-form-like helical geometry by AGO has been proposed to make productive collisions with targets more likely, because it reduces the entropic cost inherent to base pairing (Parker et al, 2009); but previous ensemble studies could only indirectly assess the rate of association (k on ) (Deerberg et al, 2013;Wee et al, 2012). Single-molecule studies now reveal that AGO proteins accelerate target RNA binding by up to 250-fold, when compared to the kinetics of protein-free nucleic acids (Salomon et al, 2015;Chandradoss et al, 2015;Jo et al, 2015). Target binding is initiated by interactions involving as little as three nucleotides within nt 2 to 5 of the guide, with increasing importance toward the 5 0 end of the guide (Salomon et al, 2015;Chandradoss et al, 2015).…”

“…These problems can be overcome by the use of biophysical approaches that trace the motion of individual molecules at biologically relevant resolution (sub-nanometer) and timescale (microseconds to minutes) (Joo et al, 2008). Four recent studies employed fluorescently labeled guide and target nucleic acid molecules by total internal reflection fluorescence (TIRF) microscopy in order to monitor the process of nucleic acid hybridization in the context of mammalian, fly, and bacterial AGO proteins (for an overview of experimental setups, see Figure 1), providing remarkably detailed insights into the mechanisms of RNAi-related processes (Salomon et al, 2015;Chandradoss et al, 2015;Jo et al, 2015;Yao et al, 2015).…”

Probing molecular choreography through single-molecule biochemistry

“…Thermodynamically, excluded volume and ligand depletion effects impact on the free energies of intermolecular interactions 20--22 and altered mobility and anomalous diffusion change the rates at which molecules find and interact with each other 23,24 . Recent single--molecule fluorescence studies have started to highlight the role simple crowding agents such as PEGs can play in stabilizing conformational states 25 and binding lifetimes 26 A quarter of human genes code for transmembrane proteins 27 and they represent targets for more than half of all drugs 28 . The use of in vitro single--molecule fluorescence methods to study membrane proteins has only recently gained traction, in part due to the many technical challenges in production and handling of membrane proteins, the requirement often for specific labeling and the ability to specifically immobilize the proteins on a surface.…”