RESUMEN
Co-evolution between hosts' and parasites' genomes shapes diverse pathways of acquired immunity based on silencing small (s)RNAs. In plants, sRNAs cause heterochromatinization, sequence degeneration, and, ultimately, loss of autonomy of most transposable elements (TEs). Recognition of newly invasive plant TEs, by contrast, involves an innate antiviral-like silencing response. To investigate this response's activation, we studied the single-copy element EVADÉ (EVD), one of few representatives of the large Ty1/Copia family able to proliferate in Arabidopsis when epigenetically reactivated. In Ty1/Copia elements, a short subgenomic mRNA (shGAG) provides the necessary excess of structural GAG protein over the catalytic components encoded by the full-length genomic flGAG-POL. We show here that the predominant cytosolic distribution of shGAG strongly favors its translation over mostly nuclear flGAG-POL. During this process, an unusually intense ribosomal stalling event coincides with mRNA breakage yielding unconventional 5'OH RNA fragments that evade RNA quality control. The starting point of sRNA production by RNA-DEPENDENT-RNA-POLYMERASE-6 (RDR6), exclusively on shGAG, occurs precisely at this breakage point. This hitherto-unrecognized "translation-dependent silencing" (TdS) is independent of codon usage or GC content and is not observed on TE remnants populating the Arabidopsis genome, consistent with their poor association, if any, with polysomes. We propose that TdS forms a primal defense against EVD de novo invasions that underlies its associated sRNA pattern.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Elementos Transponibles de ADN/genética , Regulación de la Expresión Génica de las Plantas , ARN Interferente Pequeño/genéticaRESUMEN
Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.
Asunto(s)
Proteínas Argonautas/metabolismo , Cromatografía Liquida/métodos , ARN Interferente Pequeño/aislamiento & purificación , Complejo Silenciador Inducido por ARN/química , Animales , Resinas de Intercambio Aniónico , Proteínas Argonautas/aislamiento & purificación , Línea Celular Tumoral , Biblioteca de Genes , Ratones , Ratones Endogámicos C57BL , Polinucleótido 5'-Hidroxil-Quinasa , ARN de Hongos/aislamiento & purificación , ARN de Helminto/aislamiento & purificación , ARN Neoplásico/aislamiento & purificación , ARN de Planta/aislamiento & purificación , ARN Protozoario/aislamiento & purificación , ARN Interferente Pequeño/sangre , ARN Interferente Pequeño/metabolismo , Sefarosa , Dióxido de Silicio , UltracentrifugaciónRESUMEN
Double-stranded RNA (dsRNA) pesticides are a new generation of crop protectants that interfere with protein expression in targeted pest insects by a cellular mechanism called RNA interference (RNAi). The ecological risk assessment of these emerging pesticides necessitates an understanding of the fate of dsRNA molecules in receiving environments, among which agricultural soils are most important. We herein present an experimental approach using phosphorus-32 (32P)-radiolabeled dsRNA that allows studying key fate processes of dsRNA in soils with unprecedented sensitivity. This approach resolves previous analytical challenges in quantifying unlabeled dsRNA and its degradation products in soils. We demonstrate that 32P-dsRNA and its degradation products are quantifiable at concentrations as low as a few nanograms of dsRNA per gram of soil by both Cerenkov counting (to quantify total 32P-activity) and by polyacrylamide gel electrophoresis followed by phosphorimaging (to detect intact 32P-dsRNA and its 32P-containing degradation products). We show that dsRNA molecules added to soil suspensions undergo adsorption to soil particle surfaces, degradation in solution, and potential uptake by soil microorganisms. The results of this work on dsRNA adsorption and degradation advance a process-based understanding of the fate of dsRNA in soils and will inform ecological risk assessments of emerging dsRNA pesticides.