Clarifying mammalian RISC assembly in vitro

<p>Abstract</p> <p>Background</p> <p>Argonaute, the core component of the RNA induced silencing complex (RISC), binds to mature miRNAs and regulates gene expression at transcriptional or post-transcriptional level. We recently reported that Argonaute 2 (Ago2) also assembles into complexes with miRNA precursors (pre-miRNAs). These Ago2:pre-miRNA complexes are catalytically active <it>in vitro </it>and constitute non-canonical RISCs.</p> <p>Results</p> <p>The use of pre-miRNAs as guides by Ago2 bypasses Dicer activity and complicates <it>in vitro </it>RISC reconstitution. In this work, we characterized Ago2:pre-miRNA complexes and identified RNAs that are targeted by miRNAs but not their corresponding pre-miRNAs. Using these target RNAs we were able to recapitulate <it>in vitro </it>pre-miRNA processing and canonical RISC loading, and define the minimal factors required for these processes.</p> <p>Conclusions</p> <p>Our results indicate that Ago2 and Dicer are sufficient for processing and loading of miRNAs into RISC. Furthermore, our studies suggest that Ago2 binds primarily to the 5'- and alternatively, to the 3'-end of select pre-miRNAs.</p

The Identity of Rubus pekinensis Focke and R. crataegifolius Bunge (Rosaceae)

A critical examination of specimens, with literature and a field survey have shown that Rubus pekinensis is conspecific with R. crataegifolius. Their morphological variations range can be defined as: leaves at the base may be ovate, suborbicular, narrowly ovate, entire, at the middle, ovate, narrowly ovate, oblong-lanceolate, palmately 3-lobed or 5-lobed and at the top, ovate, lanceolate, entire or 3-lobed; flowers solitary in the axillae or several flowers clustered at the terminal of branchlets or formed into short racemes. Therefore, we treat the former species as a synonym for the latter one

A novel fabrication technique for three-dimensional concave nanolens arrays

A novel facile technique is proposed for fabricating three-dimensional (3D) concave nanolens arrays on a silicon substrate. The technique leverages an inherent characteristic of the polymethyl methacrylate (PMMA) resist during inductively coupled plasma (ICP) etching. The tendency for plasma ions to accumulate at the edge of the PMMA resist helps create a local electric field that causes the ions to etch the sidewall of the PMMA resist. This process progressively increases the uncovered area, resulting in a graded etched depth or a concave structure in the substrate. In addition, using a given ICP etching recipe, the time required for a PMMA resist to be removed by sidewall etching is determined by its width. The use of PMMA resist of different widths enables one to achieve structures of varying etched depths and thus a 3D lens array. Optical characteristics of the fabricated nanolens were simulated using the FDTD (Finite-difference time-domain) method, and focal lengths ranging from 150 nm to 420 nm were obtained. This type of nanolens is very useful in ultraviolet optical devices and CMOS image sensors.Published versio

Low-Pollution and Controllable Selective-Area Deposition of a CdS Buffering Layer on CIGS Solar Cells by a Photochemical Technique

A chemical-bath deposition method has recently been applied for the industrial deposition of CdS buffer layers in high-efficiency Cu­(In, Ga)­Se₂ (CIGS) solar cells; however, its massive raw material waste and heavy pollution have also hindered its long-range industrialization. In this study, a type of low-pollution and controllable selective-area deposition of CdS thin films on cells was proposed and conducted by a photochemical deposition (PCD) technique using an aqueous solution containing S₂O₃^2–, SO₃^2–, and Cd²⁺. The as-deposited films are low-crystallinity, uniform, and compact with thicknesses of 30–50 nm. Moreover, the depositions of CdS thin films were further investigated by tuning the deposition time, absorption of cadmium ions, sulfur concentration, and light intensity. Additionally, an ion-by-ion mechanism was proposed for the growth of CdS thin films by a PCD technique. Furthermore, the optimal CdS thin layer was applied in CIGS solar cells, which showed a high efficiency of 10.45%. This research would give new insight into the efficient deposition of CdS thin films on solar cells with low pollution

Unique 5′-P recognition and basis for dG:dGTP misincorporation of ASFV DNA polymerase X

<div><p>African swine fever virus (ASFV) can cause highly lethal disease in pigs and is becoming a global threat. ASFV DNA Polymerase X (<i>Asfv</i>PolX) is the most distinctive DNA polymerase identified to date; it lacks two DNA-binding domains (the thumb domain and 8-KD domain) conserved in the homologous proteins. <i>Asfv</i>PolX catalyzes the gap-filling reaction during the DNA repair process of the ASFV virus genome; it is highly error prone and plays an important role during the strategic mutagenesis of the viral genome. The structural basis underlying the natural substrate binding and the most frequent dG:dGTP misincorporation of <i>Asfv</i>PolX remain poorly understood. Here, we report eight <i>Asfv</i>PolX complex structures; our structures demonstrate that <i>Asfv</i>PolX has one unique 5′-phosphate (5′-P) binding pocket, which can favor the productive catalytic complex assembly and enhance the dGTP misincorporation efficiency. In combination with mutagenesis and in vitro catalytic assays, our study also reveals the functional roles of the platform His115-Arg127 and the hydrophobic residues Val120 and Leu123 in dG:dGTP misincorporation and can provide information for rational drug design to help combat ASFV in the future.</p></div

5′-P of downstream oligo facilitates the productive complex assembly.

<p>(<b>A</b>) Sequence of 1nt-gap DNA4 and the overall structure of <i>Asfv</i>PolX:1nt-gap DNA4 complex. <i>Asfv</i>PolX is shown as a cartoon with the palm and finger domains colored in cyan and white, respectively. The template strand, primer, and downstream oligo are shown as stick with the C atoms colored in green, yellow, and yellow, respectively. The template residue (G8) is indicated with arrow. (<b>B</b>) Sequences of 2nt-gap(P) DNA5 and 1nt-gap(P) DNA5. (<b>C</b>) Overall structure of <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP. <i>Asfv</i>PolX is shown as cartoon with palm and finger domains colored in cyan and white, respectively. DNA is shown as sticks with the C atoms colored in yellow, green, and green, for the template strand, primer, and downstream oligo, respectively. dGTP is also shown as sticks, Mn²⁺ ions are shown as red spheres.</p

The impacts of the H115-Arg127 platform on the dG:dGTP misincorporation.

<p>The dC:dGTP and dG:dGTP base pairs observed in (<b>A</b>) <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP structure and (<b>B</b>) <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP structure, respectively. The 2F_o-F_c maps are contoured at 1.5 σ level. (<b>C</b>) Quantification and comparison of in vitro dG:dGTP misincorporation assay catalyzed by WT <i>Asfv</i>PolX, H115D, H115E, H115F, R127A, and H115F/R127A mutants (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). The data represent the mean of three independent experiments with SD values indicated by error bars. The dG:dGTP and dT:ddA base pairs observed at the insertion and postinsertion sites of (<b>D</b>) H115F/R127A:1nt-gap(P) DNA6:dGTP and (<b>E</b>) H115F:1nt-gap(P) DNA6:dGTP, respectively. (<b>F</b>) Structural comparison showing the conformational differences between <i>Asfv</i>PolX:1nt-DNA(P) DNA6:dGTP and H115F:1nt-gap(P) DNA6:dGTP. For clarity, the insertion site dG:dGTP base pairs and the <i>Asfv</i>PolX protein in <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP structure are omitted. The C atoms of Phe115, Arg127, and the postinsertion site dT:ddA of H115F:1nt-gap(P) DNA6:dGTP are colored green in both (<b>E</b>) and (<b>F</b>), whereas, the C atoms are colored white for His115, Arg127, and for the postinsertion site dT:ddA of <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP in (<b>F</b>).</p

Val120 and Leu123 affect dNTP binding and dG:dGTP misincorporation.

<p>Interactions between Val120 and the nucleobase of dG observed in (<b>A</b>) the <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP and (<b>B</b>) the <i>Asfv</i>PolX:1nt-gap(P) DNA5:dGTP structures, respectively. <i>Asfv</i>PolX is shown as a cartoon in white. dG, dGTP, His115, and ₁₁₇TGPV₁₂₀ regions are shown as sticks in atomic colors (C, magenta; N, blue; O, red; P, orange). The 2F_o-F_c map (contoured at 1.5 σ level) is shown as cyan mesh. (<b>C</b>) Hydrophobic interaction between Leu123 and the nucleobase of dGTP observed in the <i>Asfv</i>PolX:1nt-gap(P) DNA6:dGTP structure. (<b>D</b>) ITC analysis results showing the impacts of Val120 and Leu123 on the dGTP binding (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). (<b>E</b>) Quantification and comparison of in vitro dG:dGTP misincorporation assay catalyzed by WT <i>Asfv</i>PolX, V120A, and L123A mutants (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002599#pbio.1002599.s001" target="_blank">S1 Data</a>). The data represent the mean of three independent experiments with SD values indicated by error bars.</p