Structural insights into dsRNA processing by Drosophila Dicer-2-Loqs-PD.

Small interfering RNAs (siRNAs) are the key components for RNA interference (RNAi), a conserved RNA-silencing mechanism in many eukaryotes1,2. In Drosophila, an RNase III enzyme Dicer-2 (Dcr-2), aided by its cofactor Loquacious-PD (Loqs-PD), has an important role in generating 21 bp siRNA duplexes from long double-stranded RNAs (dsRNAs)3,4. ATP hydrolysis by the helicase domain of Dcr-2 is critical to the successful processing of a long dsRNA into consecutive siRNA duplexes5,6. Here we report the cryo-electron microscopy structures of Dcr-2-Loqs-PD in the apo state and in multiple states in which it is processing a 50 bp dsRNA substrate. The structures elucidated interactions between Dcr-2 and Loqs-PD, and substantial conformational changes of Dcr-2 during a dsRNA-processing cycle. The N-terminal helicase and domain of unknown function 283 (DUF283) domains undergo conformational changes after initial dsRNA binding, forming an ATP-binding pocket and a 5'-phosphate-binding pocket. The overall conformation of Dcr-2-Loqs-PD is relatively rigid during translocating along the dsRNA in the presence of ATP, whereas the interactions between the DUF283 and RIIIDb domains prevent non-specific cleavage during translocation by blocking the access of dsRNA to the RNase active centre. Additional ATP-dependent conformational changes are required to form an active dicing state and precisely cleave the dsRNA into a 21 bp siRNA duplex as confirmed by the structure in the post-dicing state. Collectively, this study revealed the molecular mechanism for the full cycle of ATP-dependent dsRNA processing by Dcr-2-Loqs-PD.

Structures and implications of the C962R protein of African swine fever virus.

African swine fever virus (ASFV) is highly contagious and can cause lethal disease in pigs. Although it has been extensively studied in the past, no vaccine or other useful treatment against ASFV is available. The genome of ASFV encodes more than 170 proteins, but the structures and functions for the majority of the proteins remain elusive, which hindered our understanding on the life cycle of ASFV and the development of ASFV-specific inhibitors. Here, we report the structural and biochemical studies of the highly conserved C962R protein of ASFV, showing that C962R is a multidomain protein. The N-terminal AEP domain is responsible for the DNA polymerization activity, whereas the DNA unwinding activity is catalyzed by the central SF3 helicase domain. The middle PriCT2 and D5_N domains and the C-terminal Tail domain all contribute to the DNA unwinding activity of C962R. C962R preferentially works on forked DNA, and likely functions in Base-excision repair (BER) or other repair pathway in ASFV. Although it is not essential for the replication of ASFV, C962R can serve as a model and provide mechanistic insight into the replicative primase proteins from many other species, such as nitratiruptor phage NrS-1, vaccinia virus (VACV) and other viruses.

The structure of MgtE in the absence of magnesium provides new insights into channel gating.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Structural basis for guide RNA trimming by RNase D ribonuclease in Trypanosoma brucei.

Infection with kinetoplastid parasites, including Trypanosoma brucei (T. brucei), Trypanosoma cruzi (T. cruzi) and Leishmania can cause serious disease in humans. Like other kinetoplastid species, mRNAs of these disease-causing parasites must undergo posttranscriptional editing in order to be functional. mRNA editing is directed by gRNAs, a large group of small RNAs. Similar to mRNAs, gRNAs are also precisely regulated. In T. brucei, overexpression of RNase D ribonuclease (TbRND) leads to substantial reduction in the total gRNA population and subsequent inhibition of mRNA editing. However, the mechanisms regulating gRNA binding and cleavage by TbRND are not well defined. Here, we report a thorough structural study of TbRND. Besides Apo- and NMP-bound structures, we also solved one TbRND structure in complexed with single-stranded RNA. In combination with mutagenesis and in vitro cleavage assays, our structures indicated that TbRND follows the conserved two-cation-assisted mechanism in catalysis. TbRND is a unique RND member, as it contains a ZFD domain at its C-terminus. In addition to T. brucei, our studies also advanced our understanding on the potential gRNA degradation pathway in T. cruzi, Leishmania, as well for as other disease-associated parasites expressing ZFD-containing RNDs.

Structural studies reveal a ring-shaped architecture of deep-sea vent phage NrS-1 polymerase.

NrS-1 is the first known phage that can infect Epsilonproteobacteria, one of the predominant primary producers in the deep-sea hydrothermal vent ecosystems. NrS-1 polymerase is a multidomain enzyme and is one key component of the phage replisome. The N-terminal Prim/Pol and HBD domains are responsible for DNA polymerization and de novo primer synthesis activities of NrS-1 polymerase. However, the structure and function of the C-terminus (CTR) of NrS-1 polymerase are poorly understood. Here, we report two crystal structures, showing that NrS-1 CTR adopts one unique hexameric ring-shaped conformation. Although the central helicase domain of NrS-1 CTR shares structural similarity with the superfamily III helicases, the folds of the Head and Tail domains are completely novel. Via mutagenesis and in vitro biochemical analysis, we identified many residues important for the helicase and polymerization activities of NrS-1 polymerase. In addition to NrS-1 polymerase, our study may also help us identify and understand the functions of multidomain polymerases expressed by many NrS-1 related phages.

A simple method for the preparation of multi-color carbon quantum dots by using reversible regulatory color transformation.

It is known that polychromatic carbon quantum dots (CQDs) can be obtained by doping and surface modification. The layer-wise synthesis of blue and green emitting CQDs (with typical sizes between 3 and 6 nm) is described here by adding oxalic acid and by introducing polycarboxy groups. By changing the external environment, the emission of CQDs can be adjusted in the blue-green spectral region (469-527 nm) under photoexcitation at 405 nm. The findings presented here provide new directions for the reversible regulatory transformation of polychromatic CQDs. The luminescence also is affected by a variety of conditions such as surface defects, degree of polymerization, polarity of external solutions, and hydrogen bonding which is studied in some detail. Graphical abstract Schematic presentation of the blue-green fluorescent transformation of the green carbon quantum dots(G-CQDs) to blue carbon quantum dots(B-CQDs).

Structural insight into the role of VAL1 B3 domain for targeting to FLC locus in Arabidopsis thaliana.

Vernalization is a pivotal stage for some plants involving many epigenetic changes during cold exposure. In Arabidopsis, an essential step in vernalization for further flowering is successful silence the potent floral repressor Flowering Locus C (FLC) by repressing histone mark. AtVal1 is a multi-function protein containing five domains that participate into many recognition processes and is validated to recruit the repress histone modifier PHD-PRC2 complex and interact with components of the ASAP complex target to the FLC nucleation region through recognizing a cis element known as CME (cold memory element) by its plant-specific B3 domain. Here, we determine the crystal structure of the B3 domain in complex with Sph/RY motif in CME. Our structural analysis reveals the specific DNA recognition by B3 domain, combined with our in vitro experiments, we provide the structural insight into the important implication of AtVAL1-B3 domain in flowering process.

Structural insights into the specific recognition of DSR by the YTH domain containing protein Mmi1.

Meiosis is one of the most dramatic differentiation programs accompanied by a striking change in gene expression profiles in fission yeast Schizosaccharomyces pombe. Whereas a number of meiosis-specific transcripts are expressed untimely in mitotic cells, and the entry of meiosis will be blocked as the accumulation of meiosis-specific mRNAs in the mitotic cells. A YTH domain containing protein Mmi1 was identified as a pivotal effector in a post-transcriptional event termed selective elimination of meiosis-specific mRNAs. Mmi1 can recognize and bind a class of meiosis-specific transcripts expressed inappropriately in mitotic cells, which all contain a conservative region called DSR, as a mark to remove them in cooperation with nuclear exosomes. Here we report the 1.6 Å resolution crystal structure of the Mmi1-YTH domain in complex with a high consensus hexanucleotide motif, which is multiple copied in the DSR region. Our structure observations, supported by site-directed mutations of key residues illustrate the mechanism for specific recognition of DSR-RNA by Mmi1. Moreover, different from other YTH domain family proteins, Mmi1-YTH domain has a distinctive RNA-binding properties although it has a similar fold as other ones.

Amorphous Gallium Oxide-Based Crosstalk-Suppressing Solar-Blind Imaging Array Prepared by One-Step Method.

The solar-blind ultraviolet band presents a unique opportunity for low-background-noise detection due to limited atmospheric light transmission. Especially, with the ability to obtain size, shape and position information on the objects, the solar-blind image sensors are receiving increasing attention. However, because the inhibition of the crosstalk in crossbar arrays induces the complexity of the preparation process, rarely are applicable solar-blind UV imaging arrays reported. This work prepared an amorphous gallium oxide (a-Ga2O3)-based diode with a remarkable rectifier ratio of up to 106. Then an imaging array was prepared by one-step method in that the diodes working as the detection elements and the switching elements inhibiting crosstalk were simultaneously deposited. Moreover, a 2 × 2 crossbar array clearly demonstrates the inhibition of the crosstalk. This work presents a simple and cost-effective method for preparing applicable solar-blind imaging arrays that inhibits crosstalk, promoting the practical application of the Ga2O3-based solar-blind UV detectors.

Air-Stable Self-Driven UV Photodetectors on Controllable Lead-Free CsCu2I3 Microwire Arrays.

The rapid evolution of the Internet of Things has engendered increased requirements for low-cost, self-powered UV photodetectors. Herein, high-performance self-driven UV photodetectors are fabricated by designing asymmetric metal-semiconductor-metal structures on the high-quality large-area CsCu2I3 microwire arrays. The asymmetrical depletion region doubles the photocurrent and response speed compared to the symmetric structure device, leading to a high responsivity of 233 mA/W to 355 nm radiation. Notably, at 0 V bias, the asymmetric device produces an open-circuit voltage of 356 mV and drives to a short-circuit current of 372 pA; meanwhile, the switch ratio (Iph/Idark) reaches up to 103, indicating its excellent potential for detecting weak light. Furthermore, the device maintains stable responses throughout 10000 UV-light switch cycles, with negligible degradation even after 90-day storage in air. Our work establishes that CsCu2I3 is a good candidate for self-powered UV detection and thoroughly demonstrates its potential as a passive device.

Structural mechanism of R2D2 and Loqs-PD synergistic modulation on DmDcr-2 oligomers.

Small interference RNAs are the key components of RNA interference, a conserved RNA silencing or viral defense mechanism in many eukaryotes. In Drosophila melanogaster, Dicer-2 (DmDcr-2)-mediated RNAi pathway plays important roles in defending against viral infections and protecting genome integrity. During the maturation of siRNAs, two cofactors can regulate DmDcr-2's functions: Loqs-PD that is required for dsRNA processing, and R2D2 that is essential for the subsequent loading of siRNAs into effector Ago2 to form RISC complexes. However, due to the lack of structural information, it is still unclear whether R2D2 and Loqs-PD affect the functions of DmDcr-2 simultaneously. Here we present several cryo-EM structures of DmDcr-2/R2D2/Loqs-PD complex bound to dsRNAs with various lengths by the Helicase domain. These structures revealed that R2D2 and Loqs-PD can bind to different regions of DmDcr-2 without interfering with each other. Furthermore, the cryo-EM results demonstrate that these complexes can form large oligomers and assemble into fibers. The formation and depolymerization of these oligomers are associated with ATP hydrolysis. These findings provide insights into the structural mechanism of DmDcr-2 and its cofactors during siRNA processing.

Polarization-Sensitive Detector Based on MoTe2/WTe2 Heterojunction for Broadband Optoelectronic Imaging.

Polarization-sensitive detectors have significant applications in modern communication and information processing. In this study. We present a polarization-sensitive detector based on a MoTe2/WTe2 heterojunction, where WTe2 forms a favorable bandgap structure with MoTe2 after forming the heterojunction. This enhances the carrier separation efficiency and photoelectric response. We successfully achieved wide spectral detection ranging from visible to near-infrared light. Specifically, under zero bias, our photodetector exhibits a responsivity (R) of 0.6 A/W and a detectivity (D*) of 3.6 × 1013 Jones for 635 nm laser illumination. Moreover, the photoswitching ratio can approach approximately 6.3 × 105. Importantly, the polarization sensitivity can reach 3.5 (5.2) at 635 (1310) nm polarized light at zero bias. This study both unveils potential for utilizing MoTe2/WTe2 heterojunctions as polarization-sensitive detectors and provides novel insights for developing high-performance optoelectronic devices.

Reversible charge-polarity control for a photo-triggered anti-ambipolar In2Se3&WSe2 heterotransistor.

Based on the charge-polarity control, a novel anti-ambipolar heterotransistor is proposed based on a special In2Se3&WSe2 van der Waals heterostructure. Unlike traditional logic transistors, our anti-ambipolar heterotransistor can treat an optical signal as an input to change its operating state, that is, with the switching of the optical signal, it shows a reversible polarity change between anti-ambipolar and P-type. Moreover, with the increase of laser power density from 0 to 4.4 mW cm-2, the current value corresponding to the anti-ambipolar peak of the device (Ipeak) shifts from 0 to 4.3 nA, and the voltage value corresponding to the anti-ambipolar peak of the device (Vpeak) can shift from -7 to -5.67 V. These phenomena demonstrate that the charge neutrality point of the anti-ambipolar heterotransistor can be selectively varied with the change of laser power density. In addition, the aforementioned device possesses a high Ion/Ioff ratio of about 104 (405 nm, 4.4 mW cm-2) at 0 V. These properties indicate that our unique anti-ambipolar In2Se3&WSe2 heterotransistor can be employed in photo-triggered inverters for future optoelectronic circuits, and it has great potential to improve the integration of overall chip circuits by implementing optoelectronic logic functions in a unit.

Activated Triplet Exciton Release for Highly Efficient Room-Temperature Phosphorescence Based on S,N-Doped Polymeric Carbon Nitride.

Polymeric carbon nitride (PCN) shows great potential applications in the areas of sustainable energy (photocatalysis and photoelectric conversion, as well as other important catalytic reactions), biosensing, biomedicine, devices, and more, but efficient phosphorescence is very scarce because of the lack of an effective synthetic method and an unsettled phosphorescent mechanism. Herein, we report a strategy to promote efficient phosphorescence to activate triplet exciton release by introduction of S and N elements. PCN could be synthesized by thiourea or urea (named S,N-PCN and N-PCN, respectively) at a relatively low reaction temperature (260 °C). S,N-PCN exhibits phosphorescence quantum yield (4.15%) higher than that (0.41%) for N-PCN. The introduction of C=S and C≡N groups in S,N-PCN networks could boost the intersystem crossing (ISC), leading to small singlet-triplet energy (ΔEST) up to more triplet exciton generation. Considering the excellent optical stability of PCN, a preliminary application of visible-light-excited PCN in advanced anticounterfeiting is proposed.

Targeted Manipulation of Cellular RNA m6A Methylation at the Single-Base Level.

Development of tools for precise manipulation of cellular mRNA m6A methylation at the base level is highly required. Here, we report an RNA-guided RNA modification strategy using a fusion protein containing deactivated nuclease Cas13b and m6A methyltransferase METTL14, namely, dCas13b-M14, which is designedly positioned in the cytoplasm. dCas13b-M14 naturally heterodimerizes with endogenous METTL3 to form a catalytic complex to methylate specific cytoplasmic mRNA under a guide RNA (gRNA). We developed assays to screen and validate the guiding specificity of varied gRNAs at single-base resolution. With an optimum combination of dCas13b-M14 and gRNAs inside cells, we have successfully tuned methylation levels of several selected mRNA m6A sites. The off-target effect was evaluated by whole transcriptome m6A sequencing, and a very minor perturbation on the methylome was revealed. Finally, we successfully utilized the editing tool to achieve de novo methylations on five selected mRNA sites. Together, this study paves the way for studying position-dependent roles of m6A methylation in a particular transcript.

Higher-Order Multiphoton Absorption Upconversion Lasing Based on ZnO/ZnMgO Multiple Quantum Wells.

In the progress of nonlinear optics, multiphoton absorption (MPA) upconversion lasing enables many vital applications in bioimaging, three-dimensional optical data storage, and photodynamic therapy. Here, efficient four-photon absorption upconversion lasing from the ZnO/ZnMgO multiple quantum wells (MQWs) at room temperature is realized. Moreover, the MPA upconversion lasing and third-harmonic generation peak generated in the MQWs under the excitation of a femtosecond (fs) laser pulse were observed concurrently, and the essential differences between each other were studied comprehensively. Compared with the ZnO film, the upconversion lasing peak of the ZnO/ZnMgO MQWs exhibits a clear blue shift. In addition, the four-photon absorption upconversion photoluminescence (PL) intensity was enhanced in the MQWs/Au nanoparticles (NPs) by the metal-localized surface plasmons (LSPs). The work paves the way for short-wavelength lasers by taking advantage of the high stability and large exciton binding energy of the MQWs' structures.

Ultralow-threshold six-photon-excited upconversion lasing in a plasmonic microcavity.

Nonlinear multiphoton absorption (MPA) upconversion lasers have critical applications in fluorescence imaging probes and biological photonics. Here, we report the realization of ultralow-threshold six-photon-excited upconversion lasing through cavity quantum electrodynamics effects in a plasmonic microcavity. The value of the Purcell factor (Fp) in hybrid whisper-gallery mode (WGM) is enhanced five-fold relative to a bare microwire (MW), which enhances the nonlinear light-matter interactions dramatically. Compared with a MW, the threshold of six-photon upconversion WGM lasing is reduced by one order magnitude due to plasmonic enhancement effects. In addition, the temperature and polarization characteristics of upconversion lasing via a plasmonic-WGM approach show a distinct evolution, different from a bare MW. This work paves the way for extreme nonlinear optics, taking advantage of the processability and high Purcell factor of plasmonic microcavities.

Strong In-Plane Optical and Electrical Anisotropies of Multilayered γ-InSe for High-Responsivity Polarization-Sensitive Photodetectors.

Recently, identifying promising new two-dimensional (2D) materials with low-symmetry structures has aroused great interest for developing monolithic polarization-sensitive photodetectors with small volume. Here, after comprehensive research of the in-plane anisotropic structure and electronic and optoelectronic properties of layered γ-InSe, a superior responsivity polarization-sensitive photodetector based on multilayer γ-InSe is constructed by a facile method. Notably, the conductance and carrier mobility of the device along the armchair direction are 11.8 and 2.35 times larger than those along the zigzag direction, respectively. Benefitting from the high efficiency of light absorption and excellent carrier mobility (221 cm2 V-1 s-1) of our multilayered γ-InSe along the armchair direction, the device exhibits a superior responsivity of 127 A/W and an external quantum efficiency (EQE) of 104%. Especially, the highest responsivity along the armchair direction of our γ-InSe polarization-sensitive photodetectors can reach as high as 78.5 A/W under polarized light. This value is much higher than those of other devices even under unpolarized light. This work not only provides an insight into the in-plane anisotropic properties of 2D layered γ-InSe but also proposes a stable and environmentally friendly candidate for anisotropic optoelectronic applications.

Cryo-EM structures of human m6A writer complexes.

N6-methyladenosine (m6A) is the most abundant ribonucleotide modification among eukaryotic messenger RNAs. The m6A "writer" consists of the catalytic subunit m6A-METTL complex (MAC) and the regulatory subunit m6A-METTL-associated complex (MACOM), the latter being essential for enzymatic activity. Here, we report the cryo-electron microscopy (cryo-EM) structures of MACOM at a 3.0-Å resolution, uncovering that WTAP and VIRMA form the core structure of MACOM and that ZC3H13 stretches the conformation by binding VIRMA. Furthermore, the 4.4-Å resolution cryo-EM map of the MACOM-MAC complex, combined with crosslinking mass spectrometry and GST pull-down analysis, elucidates a plausible model of the m6A writer complex, in which MACOM binds to MAC mainly through WTAP and METTL3 interactions. In combination with in vitro RNA substrate binding and m6A methyltransferase activity assays, our results illustrate the molecular basis of how MACOM assembles and interacts with MAC to form an active m6A writer complex.

Crystal structures and insights into precursor tRNA 5'-end processing by prokaryotic minimal protein-only RNase P.

Besides the canonical RNA-based RNase P, pre-tRNA 5'-end processing can also be catalyzed by protein-only RNase P (PRORP). To date, various PRORPs have been discovered, but the basis underlying substrate binding and cleavage by HARPs (homolog of Aquifex RNase P) remains elusive. Here, we report structural and biochemical studies of HARPs. Comparison of the apo- and pre-tRNA-complexed structures showed that HARP is able to undergo large conformational changes that facilitate pre-tRNA binding and catalytic site formation. Planctomycetes bacterium HARP exists as dimer in vitro, but gel filtration and electron microscopy analysis confirmed that HARPs from Thermococcus celer, Thermocrinis minervae and Thermocrinis ruber can assemble into larger oligomers. Structural analysis, mutagenesis and in vitro biochemical studies all supported one cooperative pre-tRNA processing mode, in which one HARP dimer binds pre-tRNA at the elbow region whereas 5'-end removal is catalyzed by the partner dimer. Our studies significantly advance our understanding on pre-tRNA processing by PRORPs.