Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

<p>Circular RNAs (circRNAs) were recently described as a novel class of cellular RNAs. Two circRNAs were reported to function as molecular sponges, sequestering specific microRNAs, thereby de-repressing target mRNAs. Due to their elevated stability in comparison to linear RNA, circRNAs may be an interesting tool in molecular medicine and biology. In this study, we provide a proof-of-principle that circRNAs can be engineered as microRNA sponges. As a model system, we used the Hepatitis C Virus (HCV), which requires cellular microRNA-122 for its life cycle. We produced artificial circRNA sponges <i>in vitro</i> that efficiently sequester microRNA-122, thereby inhibiting viral protein production in an HCV cell culture system. These circRNAs are more stable than their linear counterparts, and localize both to the cytoplasm and to the nucleus, opening up a wide range of potential applications.</p

(A-E) The fungal silencing machinery is required for efficient SIGS in distal leaf parts.

<p><b>(A,B)</b> The fungal <i>dicer-like-1</i> mutant Fg-IFA65_Δdcl-1 heavily infected barley leaves despite a prior spray-treatment with <i>CYP3</i>-dsRNA. Photographs were taken at 6 dpi. <b>(C)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts in the wild type Fg-IFA65 and the mutant Fg-IFA65_Δdcl-1 at 6 dpi in the distal, semi-systemic leaf areas. <b>(D)</b> Inhibition of <i>CYP51</i> gene expression upon <i>CYP3</i>-dsRNA treatment of axenically grown Fg-IFA65_- Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in samples treated with <i>CYP3</i>-dsRNA compared with mock-treated controls was statistically significant (*P < 0.05, **P < 0.01; Student´s t test). <b>(E-G)</b> Profiling of <i>CYP3</i>-dsRNA-derived sRNAs in axenically grown Fg-IFA65. (E) Scaffold of the 791 nt long <i>CYP3</i>-dsRNA. The fragments of <i>CYP51</i> genes are indicated. (F,G) Total sRNAs were isolated from axenically-cultured Fg-IFA65. sRNA reads of fungal sRNAs from untreated (F) and <i>CYP3</i>-dsRNA-treated (G) fungal cultures are mapped to the sequence of <i>CYP3</i>-dsRNA.</p

(A-E) The fungal silencing machinery is required for efficient SIGS in distal leaf parts.

<p><b>(A,B)</b> The fungal <i>dicer-like-1</i> mutant Fg-IFA65_Δdcl-1 heavily infected barley leaves despite a prior spray-treatment with <i>CYP3</i>-dsRNA. Photographs were taken at 6 dpi. <b>(C)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts in the wild type Fg-IFA65 and the mutant Fg-IFA65_Δdcl-1 at 6 dpi in the distal, semi-systemic leaf areas. <b>(D)</b> Inhibition of <i>CYP51</i> gene expression upon <i>CYP3</i>-dsRNA treatment of axenically grown Fg-IFA65_- Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in samples treated with <i>CYP3</i>-dsRNA compared with mock-treated controls was statistically significant (*P < 0.05, **P < 0.01; Student´s t test). <b>(E-G)</b> Profiling of <i>CYP3</i>-dsRNA-derived sRNAs in axenically grown Fg-IFA65. (E) Scaffold of the 791 nt long <i>CYP3</i>-dsRNA. The fragments of <i>CYP51</i> genes are indicated. (F,G) Total sRNAs were isolated from axenically-cultured Fg-IFA65. sRNA reads of fungal sRNAs from untreated (F) and <i>CYP3</i>-dsRNA-treated (G) fungal cultures are mapped to the sequence of <i>CYP3</i>-dsRNA.</p

(A,B) Defense-related salicylate- and jasmonate-responsive genes are not induced by <i>CYP3</i>-dsRNA.

<p>Detached second leaves of three-week-old barley were sprayed with 20 ng μL^-1 <i>CYP3</i>-dsRNA or TE (control), respectively, and 48 h later drop-inoculated with Fg-IFA65. Leaves were harvested 6 dpi and analyzed for gene expression by qPCR: <b>(A)</b> <i>Pathogenesis-related</i> 1 (<i>HvPR1</i>) and <b>(B)</b> <i>S-adenosyl-l-methionine</i>:<i>jasmonic acid carboxyl methyltransferase</i> (<i>HvJMT</i>). Both genes are highly responsive to Fg-IFA65 but not to <i>CYP3</i>-dsRNA or TE treatment. Please note that a combined treatment of <i>CYP3</i>-dsRNA followed by Fg-IFA65 48 h later also did not induce these marker genes, which shows independently that fungal development on <i>CYP3</i>-dsRNA-treated leaves is strongly inhibited.</p

(A,B) Northern gel blot analysis of <i>CYP3</i>-dsRNA and <i>CYP3</i>-dsRNA-derived siRNA accumulation in local and distal (semi-systemic) barley leaf areas.

<p><b>(A)</b> Detection of 791 nt long <i>CYP3</i>-dsRNA precursor in pooled leaf tissue from non-infected leaves using [α-32P]-dCTP labeled <i>CYP3</i>-dsRNA as probe. Local (L) and distal (semi-systemic [S]) leaf segments were sampled separately at the indicated times after spraying with <i>CYP3-</i>dsRNA. No signal was detected in samples from TE-sprayed plants. <b>(B)</b> Recording <i>CYP3</i>-dsRNA-derived small RNAs in local and distal (semi-systemic) leaf areas using [α-32P]-dCTP labeled <i>CYP3</i>-dsRNA as probe. In this experiment, small RNAs could not be detected in distal (non-sprayed) tissues. siRNA generated <i>in vitro</i> by a commercial Dicer preparation from <i>CYP3</i>-dsRNA was used as positive control. No signal was detected in samples from TE-sprayed plants. Ethidium bromide-stained rRNA served as the loading control. Signals originate from the same membrane but different exposure times.</p

(A-D) SIGS-mediated semi-systemic control of <i>Fusarium graminearum</i>.

<p><b>(A)</b> Upper parts of detached second leaves of three-week-old barley were sprayed evenly with <i>CYP3</i>-dsRNA, TE, and <i>GFP</i>-dsRNA, respectively. After 48 h, the non-inoculated, semi-systemic (distal) tissue was drop-inoculated with 2 × 10⁴ conidia mL⁻¹ of Fg-IFA65_GFP; the relative amount of fungal DNA in distal tissue, as measured by qPCR at 6 dpi, was reduced in <i>CYP3</i>-dsRNA-treated leaves. Bars represent mean values ± SDs of three independent experiments. The reduction of fungal growth on <i>CYP3</i>-dsRNA-sprayed leaves was statistically significant (**P < 0.01; Student´s t test). <b>(B)</b> Gene-specific qPCR analysis of <i>CYP51A</i>, <i>CYP51B</i>, and <i>CYP51C</i> transcripts at 6 dpi in distal leaf areas. Bars represent mean values ±SDs of three independent sample collections. The reduction in <i>CYP51</i> expression in leaves sprayed with <i>CYP3</i>-dsRNA compared with <i>GFP</i>-dsRNA-sprayed controls was statistically significant (**P < 0.01, ***P < 0.001; Student´s t test). <b>(C,D)</b> Microscopy of fungal growth at semi-systemic sites of drop-inoculation with Fg-IFA65_GFP. <b>(C)</b> Successful fungal colonization (green) on TE-sprayed leaves. Profuse hyphal growth is seen inside the cells (plasma membrane stained with RH414 is highlighted in red) <b>(D)</b> Hyphal formation is strongly reduced and confined to the inoculated leaf area on <i>CYP3</i>-dsRNA-sprayed leaves. Impaired spore germination was observed in the area around the inoculation site while the surrounding cells remained free of colonization. (IF, infection hyphae; GS, germinating spore). Photographs for C and D were taken at 6 dpi.</p

(A-C) Spray-induced gene silencing (SIGS) of <i>GFP</i> expression in <i>Fusarium graminearum</i> strain Fg-IFA65_GFP.

<p>Detached second leaves of three-week-old barley plants were locally sprayed with Tris-EDTA (TE, <b>A</b>, control) or <i>GFP</i>-dsRNA <b>(B)</b>. Forty-eight hours after spraying, distal, non-sprayed leaf segments were drop-inoculated with Fg-IFA65_GFP (20 μL of a solution containing 2 x 10⁴ conidia mL^-1). <i>GFP</i> silencing efficiency was visualized 6 dpi using confocal microscopy. <b>(C)</b> <i>GFP</i> transcripts were quantified by qPCR at 6 dpi. The reduction in fungal <i>GFP</i> expression on leaves sprayed with <i>GFP</i>-dsRNA and infected with Fg-IFA65_GFP compared with TE-sprayed controls was statistically significant (***P < 0.001; Student´s <i>t</i> test). Bars represent mean values ± SDs of three independent experiments. Scale bars represent 100 μm.</p

(A-J) Confocal laser scanning microscopy of ATTO 488-labeled <i>CYP3</i>-dsRNA_A488 in locally sprayed barley leaves.

<p><b>(A-C)</b> Detection of <i>CYP3</i>-dsRNA_A488 (green) in xylem vessels of vascular bundles 24 h after spraying. <b>(D-G)</b> Longitudinal sections reveal uptake of <i>CYP3</i>-dsRNA_A488 by cells of the phloem tissue at 24 h after spraying. SE, sieve element; CC, companion cell; SP, sieve plate; PPC, phloem parenchyma cell; MC, mesophyll cell. The red cells result from the autofluorescence of chloroplasts (F,G). <b>(H-J)</b> Leaf hair cells (trichome), stomata, germinating spores (GS) and fungal hyphae strongly accumulated <i>CYP3</i>-dsRNA_A488. Fungal hyphae (IF) are stained with chitin-specific dye WGA-Alexa Fluor 594 (red) 24 h after inoculation. EC, epidermal cells. RNA signals in germinated conidia are marked by arrow heads. Scale bars 100 μm (A-H), 20 μm (F), and 10 μm (J).</p