Abstract
Drosophila
Dicer-2 (Dcr-2) produces small interfering RNAs from long double-stranded RNAs (dsRNAs), playing an essential role in antiviral RNA interference. The dicing reaction by Dcr-2 is ...enhanced by Loquacious-PD (Loqs-PD), a dsRNA-binding protein that partners with Dcr-2. Previous biochemical analyses have proposed that Dcr-2 uses two distinct—processive or distributive—modes of cleavage by distinguishing the terminal structures of dsRNAs and that Loqs-PD alters the terminal dependence of Dcr-2. However, the direct evidence for this model is lacking, as the dynamic movement of Dcr-2 along dsRNAs has not been traced. Here, by utilizing single-molecule imaging, we show that the terminal structures of long dsRNAs and the presence or absence of Loqs-PD do not essentially change Dcr-2’s cleavage mode between processive and distributive, but rather simply affect the probability for Dcr-2 to undergo the cleavage reaction. Our results provide a refined model for how the dicing reaction by Dcr-2 is regulated.
Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after ...target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 5′ cleavage fragment from the guide 3′ supplementary region first and then the 3′ fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 3′ supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 3′ supplementary region upon target recognition.
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•The RNAi-mediated target cleavage reaction was analyzed by single-molecule imaging•The seed base paring is disproportionally stabilized throughout the reaction•Target recognition proceeds via 5′ → 3′ base-pairing propagation with proofreading•Cleaved fragments are released independently based on the sequence and stability
The RNA-induced silencing complex (RISC) mediates target cleavage in RNAi. Yao et al. show, by single-molecule imaging, that Drosophila Ago2-RISC releases the cleavage fragments, with the order influenced by the stability in each region, contrary to the unidirectional base-pairing propagation from the seed to the 3′ supplementary region upon target recognition.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Bending of cilia and flagella occurs when axonemal dynein molecules on one side of the axoneme produce force and move toward the microtubule (MT) minus end. These dyneins are then pulled back when ...the axoneme bends in the other direction, meaning oscillatory back and forth movement of dynein during repetitive bending of cilia/flagella. There are various factors that may regulate the dynein activity, e.g. the nexin-dynein regulatory complex, radial spokes, and central apparatus. In order to understand the basic mechanism of dynein’s oscillatory movement, we constructed a simple model system composed of MTs, outer-arm dyneins, and crosslinks between the MTs made of DNA origami. Electron microscopy (EM) showed pairs of parallel MTs crossbridged by patches of regularly arranged dynein molecules bound in two different orientations, depending on which of the MTs their tails bind to. The oppositely oriented dyneins are expected to produce opposing forces when the pair of MTs have the same polarity. Optical trapping experiments showed that the dynein-MT-DNA-origami complex actually oscillates back and forth after photolysis of caged ATP. Intriguingly, the complex, when held at one end, showed repetitive bending motions. The results show that a simple system composed of ensembles of oppositely oriented dyneins, MTs, and inter-MT crosslinkers, without any additional regulatory structures, has an intrinsic ability to cause oscillation and repetitive bending motions.
Cellular transport systems are sophisticated and efficient. Hence, one of the ultimate goals of nanotechnology is to design artificial transport systems rationally. However, the design principle has ...been elusive, because how motor layout affects motile activity has not been established, partially owing to the difficulty in achieving a precise layout of the motile elements. Here, we employed a DNA origami platform to evaluate the two-dimensional (2D) layout effect of kinesin motor proteins on transporter motility. We succeeded in accelerating the integration speed of the protein of interest (POI) to the DNA origami transporter by up to 700 times by introducing a positively charged poly-lysine tag (Lys-tag) into the POI (kinesin motor protein). This Lys-tag approach allowed us to construct and purify a transporter with high motor density, allowing a precise evaluation on the 2D layout effect. Our single-molecule imaging showed that the densely packed layout of kinesin decreased the run length of the transporter, although its velocity was moderately affected. These results indicate that steric hindrance is a critical parameter to be considered in the design of transport systems.
Single-molecule fluorescence imaging of DNA origami-based transporters showed shorter run lengths in dence layouts of kinesin motors.
The motor protein kinesin has two heads and walks along microtubules processively using energy derived from ATP. However, how kinesin heads are coordinated to generate processive movement remains ...elusive. Here we created a hybrid nanomachine (DNA‐kinesin) using DNA as the skeletal structure and kinesin as the functional module. Single molecule imaging of DNA‐kinesin hybrid allowed us to evaluate the effects of both connect position of the heads (N, C‐terminal or Mid position) and sub‐nanometer changes in the distance between the two heads on motility. Our results show that although the native structure of kinesin is not essential for processive movement, it is the most efficient. Furthermore, forward bias by the power stroke of the neck linker, a 13‐amino‐acid chain positioned at the C‐terminus of the head, and internal strain applied to the rear of the head through the neck linker are crucial for the processive movement. Results also show that the internal strain coordinates both heads to prevent simultaneous detachment from the microtubules. Thus, the inter‐head coordination through the neck linker facilitates long‐distance walking.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Co-translational protein folding is one of the central topics in molecular biology. In
Escherichia coli
, trigger factor (TF) is a primary chaperone that facilitates co-translational folding by ...directly interacting with nascent polypeptide chains on translating ribosomes. In this study, we applied fluorescence correlation spectroscopy (FCS), which can analyze the diffusion properties of fluorescent molecules by measuring the fluctuations of the fluorescent intensity, to investigate the interaction between TF and a nascent chain on translating ribosomes both
in vitro
and
in vivo
. The FCS analysis with a reconstituted cell-free translation system revealed that the interaction of fluorescently labeled TF with a nascent chain depended on the emergence of the nascent chain from the ribosome exit tunnel, and this interaction was not inhibited by excess amounts of other chaperones. Furthermore, the translation-dependent interaction between GFP-fused TFs and nascent chains was also observed in living
E. coli
cells. The FCS-based approach established here could be an effective method to investigate the dynamics of other ribosome-associated chaperones besides TF.
Ribavirin, a nucleic acid analog, has been employed as an antiviral agent against RNA and DNA viruses and has become the standard agent used for chronic hepatitis C in combination with ...interferon-α2a. Furthermore, the potential antitumor efficacy of ribavirin has attracted increasing interest. Recently, we demonstrated a dose-dependent antitumor effect of ribavirin for seven types of malignant glioma cell lines. However, the mechanism underlying the antitumor effect of ribavirin has not yet been fully elucidated. Therefore, the main aim of the present study was to provide further relevant data using two types of malignant glioma cell lines (U-87MG and U-138MG) with different expression of MGMT. Dotted accumulations of γH2AX were found in the nuclei and increased levels of ATM and phosphorylated ATM protein expression were also observed following ribavirin treatment (10 µM of ribavirin, clinical relevant concentration) in both the malignant glioma cells, indicating double-strand breaks as one possible mechanism underlying the antitumor effect of ribavirin. In addition, based on assessements using FACS, ribavirin treatment tended to increase the G0/G1 phase, with a time‑lapse, indicating the induction of G0/G1-phase arrest. Furthermore, an increased phosphorylated p53 and p21 protein expression was confirmed in both glioma cells. Additionally, analysis by FACS indicated that apoptosis was induced following ribavirin treatment and caspase cascade, downstream of the p53 pathway, which indicated the activation of both exogenous and endogenous apoptosis in both malignant glioma cell lines. These findings may provide an experimental basis for the clinical treatment of glioblastomas with ribavirin.
Visualization and monitoring of endogenous mRNA in the cytoplasm of living cells promises a significant comprehension of refined post-transcriptional regulation. Fluorescently labeled linear ...antisense oligonucleotides can bind to natural mRNA in a sequence-specific way and, therefore, provide a powerful tool in probing endogenous mRNA. Here, we investigated the feasibility of using linear antisense probes to monitor the variable and dynamic expression of endogenous cytoplasmic mRNAs. Two linear antisense 2'-O-methyl RNA probes, which have different interactive fluorophores at the 5'-end of one probe and at the 3'-end of the other, were used to allow fluorescence resonance energy transfer (FRET) upon hybridization to the target mRNA. By characterizing the formation of the probe-mRNA hybrids in living cells, we found that the probe composition and concentration are crucial parameters in the visualization of endogenous mRNA with high specificity. Furthermore, rapid hybridization (within 1 min) of the linear antisense probe enabled us to visualize dynamic processes of endogenous c-fos mRNA, such as fast elevation of levels after gene induction and the localization of c-fos mRNA in stress granules in response to cellular stress. Thus, our approach provides a basis for real time monitoring of endogenous cytoplasmic mRNA in living cells.
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