Chirlmin Joo

A Dynamic Search Process Underlies MicroRNA Targeting

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 the 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.

A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.

Single-molecule FRET with total internal reflection microscopy

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. This article focuses primarily on smFRET based on total internal reflection (TIR) microscopy. It begins with discussions of dye choice and labeling of nucleic acids and proteins. These are followed by information on surface preparation and data acquisition. Various methods of data analysis are then presented, as is information on setting up TIR microscopy, both the objective and the prism types.

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single‐molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre‐miRNAs) in the absence of Lin28. We find that the overhang of a pre‐miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre‐miRNAs, which have a 1‐nt 3′ overhang, TUT7 restores the canonical end structure (2‐nt 3′ overhang) through mono‐uridylation, thereby promoting miRNA biogenesis. For pre‐miRNAs where the 3′ end is further recessed into the stem (as in 3′ trimmed pre‐miRNAs), TUT7 generates an oligo‐U tail that leads to degradation. In contrast to Lin28‐stimulated oligo‐uridylation, which is processive, a distributive mode is employed by TUT7 for both mono‐ and oligo‐uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7‐RNA interaction, thus explaining how TUT7 differentiates pre‐miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre‐miRNAs.

Surface Passivation for Single-molecule Protein Studies

Single-molecule fluorescence spectroscopy has proven to be instrumental in understanding a wide range of biological phenomena at the nanoscale. Important examples of what this technique can yield to biological sciences are the mechanistic insights on protein-protein and protein-nucleic acid interactions. When interactions of proteins are probed at the single-molecule level, the proteins or their substrates are often immobilized on a glass surface, which allows for a long-term observation. This immobilization scheme may introduce unwanted surface artifacts. Therefore, it is essential to passivate the glass surface to make it inert. Surface coating using polyethylene glycol (PEG) stands out for its high performance in preventing proteins from non-specifically interacting with a glass surface. However, the polymer coating procedure is difficult, due to the complication arising from a series of surface treatments and the stringent requirement that a surface needs to be free of any fluorescent molecules at the end of the procedure. Here, we provide a robust protocol with step-by-step instructions. It covers surface cleaning including piranha etching, surface functionalization with amine groups, and finally PEG coating. To obtain a high density of a PEG layer, we introduce a new strategy of treating the surface with PEG molecules over two rounds, which remarkably improves the quality of passivation. We provide representative results as well as practical advice for each critical step so that anyone can achieve the high quality surface passivation.

Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets

MicroRNAs (miRNAs) direct post-transcriptional regulation of human genes by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. An enigmatic feature of many conserved mammalian miRNA target sites is that an adenosine (A) nucleotide opposite miRNA nucleotide-1 confers enhanced target repression independently of base pairing potential to the miRNA. In this study, we show that human Argonaute2 (Ago2) possesses a solvated surface pocket that specifically binds adenine nucleobases in the 1 position (t1) of target RNAs. t1A nucleotides are recognized indirectly through a hydrogen-bonding network of water molecules that preferentially interacts with the N6 amine on adenine. t1A nucleotides are not utilized during the initial binding of Ago2 to its target, but instead function by increasing the dwell time on target RNA. We also show that N6 adenosine methylation blocks t1A recognition, revealing a possible mechanism for modulation of miRNA target site potency. DOI: http://dx.doi.org/10.7554/eLife.07646.001

Preparing sample chambers for single-molecule FRET

Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. To study the conformational changes of individual molecules over extended time periods, molecules must be localized in space. This protocol describes the preparation of sample chambers with either bovine serum albumin (BSA)- or polyethylene glycol (PEG)-coated slides to which single molecules can be tethered for use in FRET studies.

Bringing single-molecule spectroscopy to macromolecular protein complexes

Single-molecule fluorescence spectroscopy offers real-time, nanometer-resolution information. Over the past two decades, this emerging single-molecule technique has been rapidly adopted to investigate the structural dynamics and biological functions of proteins. Despite this remarkable achievement, single-molecule fluorescence techniques must be extended to macromolecular protein complexes that are physiologically more relevant for functional studies. In this review, we present recent major breakthroughs for investigating protein complexes within cell extracts using single-molecule fluorescence. We outline the challenges, future prospects and potential applications of these new single-molecule fluorescence techniques in biological and clinical research.

Cooperative conformational transitions keep reca filament active during ATPase cycle

The active, stretched conformation of the RecA filament bound to single-stranded DNA is required for homologous recombination. During this process, the RecA filament mediates the homology search and base pair exchange with a complementary sequence. Subsequently, the RecA filament dissociates from DNA upon reaction completion. ATP binding and hydrolysis is critical throughout these processes. Little is known about the timescale, order of conversion between different cofactor bound forms during ATP hydrolysis, and the associated changes in filament conformation. We used single-molecule fluorescence techniques to investigate how ATP hydrolysis is coupled with filament dynamics. For the first time, we observed real-time cooperative structural changes within the RecA filament. This cooperativity between neighboring monomers provides a time window for nucleotide cofactor exchange, which keeps the filament in the active conformation amidst continuous cycles of ATP hydrolysis.

Why Argonaute is needed to make microRNA target search fast and reliable.

MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss recent single-molecule and high-resolution structural data in the light of theoretical work appropriated from the study of transcription-factor search. We suggest that experimentally observed internal states of the Ago-miRNA search complex may have the explicit biological function of speeding up search while maintaining specificity.