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. 2010 Oct 12;107(41):17466-73.
doi: 10.1073/pnas.1012891107. Epub 2010 Sep 24.

Global Effects of the Small RNA Biogenesis Machinery on the Arabidopsis Thaliana Transcriptome

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Global Effects of the Small RNA Biogenesis Machinery on the Arabidopsis Thaliana Transcriptome

Sascha Laubinger et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

In Arabidopsis thaliana, four different dicer-like (DCL) proteins have distinct but partially overlapping functions in the biogenesis of microRNAs (miRNAs) and siRNAs from longer, noncoding precursor RNAs. To analyze the impact of different components of the small RNA biogenesis machinery on the transcriptome, we subjected dcl and other mutants impaired in small RNA biogenesis to whole-genome tiling array analysis. We compared both protein-coding genes and noncoding transcripts, including most pri-miRNAs, in two tissues and several stress conditions. Our analysis revealed a surprising number of common targets in dcl1 and dcl2 dcl3 dcl4 triple mutants. Furthermore, our results suggest that the DCL1 is not only involved in miRNA action but also contributes to silencing of a subset of transposons, apparently through an effect on DNA methylation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Global gene expression profiles in miRNA biogenesis mutants determined with tiling arrays. (A) Comparison of annotated genes up-regulated in dcl1, hyl1, and se mutants. (B) Heat map of pri-miRNA expression. (C) Comparison of three pri-miRNAs that are highly expressed in both wild-type plants and mutants with a canonical pri-miRNA, MIR319a, which accumulates only in miRNA-processing mutants. Secondary structure predictions below, indicating extensive doublestrandedness of the precursors, are from the Arabidopsis Small RNA Project (ASRP) (53). (D) Tiling array hybridization intensities for two miR172 precursors are averaged across three biological replicates.
Fig. 2.
Fig. 2.
Expression of pri-miRNAs and genes encoding miRNA biogenesis factors in stress-treated dcl1 mutants. (A) Heat map of pri-miRNA expression in dcl1 mutant seedlings. (B) Examples of pri-miRNAs specifically up-regulated in response to heat stress. (C) Expression changes of genes involved in miRNA processing and action (DCL1, HYL1, SE, HEN1, HASTY, and AGO1) and genes encoding a set of related RNA exonucleases (SDN1, At2g48100, At5g61390, and At1g74390).
Fig. 3.
Fig. 3.
Transcriptionally active regions (TARs) that specifically appear in miRNA-processing mutants. (A) Fractions of intergenic TARs among those that were significantly induced or repressed relative to wild type (Mann–Whitney U test, α ≤ 5%). (B) Overlap of total length (in kilobases) among unannotated TARs with significantly higher expression in dcl1, hyl1, and se compared to wild type. Fractions of unique TARs are indicated in gray. (C) Hybridization intensities on tiling arrays for an unannotated TAR detected exclusively in hyl1 mutants. An unannotated expressed sequence tag clone is shown in green.
Fig. 4.
Fig. 4.
Many TARs likely identify unannotated portions of pri-miRNAs. (A) Distances of unannotated TARs with induced expression in mutant inflorescences from annotated miRNA genes. (B) RT-PCR analysis of pri-miRNA865. Primers for the reaction on the right spanned splice junctions. (C) Quantitative RT-PCR analysis of pri-miRNA865. Error bars indicate the range of two independent biological experiments.
Fig. 5.
Fig. 5.
Analysis of two helitron-type transposons. (A) Comparison of tiling array expression analysis and small RNA profiles from ref. . (B) RT-PCR analysis. (C and D) Quantitative RT-PCR analysis. (E and F) Analysis of DNA methylation using digest of genomic DNA with methylation-sensitive restriction enzymes followed by PCR (E) or sequencing of bisulfate-treated genomic DNA (F). For the latter, at least 30 clones were sequenced for each genotype. Sequence contexts are shown above.
Fig. 6.
Fig. 6.
Comparison of dcl1 and dcl2 dcl3 dcl4 mutants. (A) Overlap in annotated genes that were up-regulated relative to wild type at a false-discovery rate (FDR) of 0.1. (B) Fraction of intergenic TARs among all TARs that were significantly induced or repressed relative to wild type (Mann–Whitney U test, α ≤ 5%). (C) Overlap of total length (in kilobases) among TARs that are unannotated and induced in dcl1 and dcl2 dcl3 dcl4 mutants. (D) Fractions of TARs that overlap with annotated transposable elements among TARs that were induced relative to wild type.

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