Integrated genome-scale analysis and northern blot detection of retrotransposon siRNAs across plant species

Authors: BOHRER, MARCEL - Université De Strasbourg: Accueil; RYMEN, BART - Université De Strasbourg: Accueil; HIMBER, CHRISTOPHE - Université De Strasbourg: Accueil; GERBAUD, AUDE - Université De Strasbourg: Accueil; PFLIEGER, DAVID - Université De Strasbourg: Accueil; L Chingcuanco, Debbie; CARWRIGHT, AMY - US Department Of Energy; VOGEL, JOHN - US Department Of Energy; SIBOUT, RICHARD - Inra, Génétique Animale Et Biologie Intégrative , Jouy-En-josas, France; BLEVINS, TODD - Université De Strasbourg: Accueil

Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 1/10/2020
Publication Date: 1/10/2020
Citation: Bohrer, M., Rymen, B., Himber, C., Gerbaud, A., Pflieger, D., Chingcuanco, D.L., Carwright, A., Vogel, J., Sibout, R., Blevins, T. 2020. Integrated genome-scale analysis and northern blot detection of retrotransposon siRNAs across plant species. Heinlein, H. editor. RNA Tagging Methods and Protocols Methods in Molecular Biology 2166., New York, N.Y., Springer Science + Business Media, LLC., p.387-411. https://doi.org/10.1007/978-1-0716-0712-1_23.
DOI: https://doi.org/10.1007/978-1-0716-0712-1_23

Interpretive Summary: The article is part of a book “that provides a compendium of state-of-the-art methods for the labeling, detection, and purification of RNA and RNA-protein complexes and thereby constitutes an important toolbox for researchers interested in understanding the complex roles of RNA molecules in development, signaling, and disease. Beginning with a section on in situ detection of RNA molecules using FISH techniques, the volume continues with parts exploring in vivo imaging of RNA transport and localization, imaging and analysis of RNA uptake and transport between cells, identification and analysis of RNA-binding proteins, guide RNAs in genome editing, as well as other specific analytical techniques”. Authoritative and practical, RNA Tagging: Methods and Protocols serves as a vital reference for researchers looking to further the increasingly important research in RNA biology.

Technical Abstract: Cells have sophisticated RNA-directed mechanisms to regulate genes, destroy viruses or silence transposable elements (TEs). In terrestrial plants, a specialized non-coding RNA machinery involving RNA polymerase IV (Pol IV) and small interfering RNAs (siRNAs) targets DNA methylation and silencing to TEs. Here, we present a bioinformatics protocol for annotating and quantifying siRNAs that derive from long-terminal repeat (LTR) retrotransposons. The approach was validated using small RNA northern blot analyses, comparing the species Arabidopsis thaliana and Brachypodium distachyon. To assist hybridization probe design, we configured a genome browser to show small RNA-seq mappings in distinct colors and shades according to their nucleotide lengths and abundances, respectively. Samples from wild-type and pol IV mutant plants, cross-species negative controls, and a conserved microRNA control validated the detected siRNA signals, confirming their origin from specific TEs and their Pol IV-dependent biogenesis. Moreover, an optimized labeling method yielded probes thatcould detect low abundance siRNAs from B. distachyon TEs. The integration of de novo TE annotation, small RNA-seq profiling and northern blotting, as outlined here, will facilitate the comparative genomic analysis of RNA silencing in crop plants and non-model species.