Chapter 23
Integrated Genome-Scale Analysis and Northern Blot
Detection of Retrotransposon siRNAs Across Plant Species
Marcel Bo ¨ hrer, Bart Rymen, Christophe Himber, Aude Gerbaud,
David Pflieger, Debbie Laudencia-Chingcuanco, Amy Cartwright,
John Vogel, Richard Sibout, and Todd Blevins
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 that could 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.
Key words siRNAs, Northern blotting, RNA polymerase IV (Pol IV), Long terminal repeat (LTR)
retrotransposon, Arabidopsis thaliana, Brachypodium distachyon
1 Introduction
RNA interference is a form of sequence-specific gene silencing in
eukaryotes: double-stranded RNAs (dsRNAs), arising in cells or of
exogenous origin, are diced into small interfering RNAs (siRNAs),
which guide the cleavage of complementary RNA targets leading to
transcript degradation [1, 2]. Key evidence for RNA interference
was documented in plants in the early 1990s, culminating with the
northern blot detection of siRNAs from transgenes and viruses
undergoing silencing [2]. Around the beginning of the
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_23, © Springer Science+Business Media, LLC, part of Springer Nature 2020
387
Integrated Genome-Scale Analysis and Northern Blot
Detection of Retrotransposon siRNAs Across Plant Species
Marcel Bo ¨ hrer, Bart Rymen, Christophe Himber, Aude Gerbaud,
David Pflieger, Debbie Laudencia-Chingcuanco, Amy Cartwright,
John Vogel, Richard Sibout, and Todd Blevins
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 that could 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.
Key words siRNAs, Northern blotting, RNA polymerase IV (Pol IV), Long terminal repeat (LTR)
retrotransposon, Arabidopsis thaliana, Brachypodium distachyon
1 Introduction
RNA interference is a form of sequence-specific gene silencing in
eukaryotes: double-stranded RNAs (dsRNAs), arising in cells or of
exogenous origin, are diced into small interfering RNAs (siRNAs),
which guide the cleavage of complementary RNA targets leading to
transcript degradation [1, 2]. Key evidence for RNA interference
was documented in plants in the early 1990s, culminating with the
northern blot detection of siRNAs from transgenes and viruses
undergoing silencing [2]. Around the beginning of the
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_23, © Springer Science+Business Media, LLC, part of Springer Nature 2020
387