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. 2013 Apr;41(8):4699-708.
doi: 10.1093/nar/gkt152. Epub 2013 Mar 12.

Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants

Ana Beatriz Moreno  1 , Angel Emilio Martínez de Alba, Florian Bardou, Martin D Crespi, Hervé Vaucheret, Alexis Maizel, Allison C Mallory
Affiliations

Cytoplasmic and nuclear quality control and turnover of single-stranded RNA modulate post-transcriptional gene silencing in plants

Ana Beatriz Moreno et al. Nucleic Acids Res. 2013 Apr.

Abstract

Eukaryotic RNA quality control (RQC) uses both endonucleolytic and exonucleolytic degradation to eliminate dysfunctional RNAs. In addition, endogenous and exogenous RNAs are degraded through post-transcriptional gene silencing (PTGS), which is triggered by the production of double-stranded (ds)RNAs and proceeds through short-interfering (si)RNA-directed ARGONAUTE-mediated endonucleolytic cleavage. Compromising cytoplasmic or nuclear 5'-3' exoribonuclease function enhances sense-transgene (S)-PTGS in Arabidopsis, suggesting that these pathways compete for similar RNA substrates. Here, we show that impairing nonsense-mediated decay, deadenylation or exosome activity enhanced S-PTGS, which requires host RNA-dependent RNA polymerase 6 (RDR6/SGS2/SDE1) and SUPPRESSOR OF GENE SILENCING 3 (SGS3) for the transformation of single-stranded RNA into dsRNA to trigger PTGS. However, these RQC mutations had no effect on inverted-repeat-PTGS, which directly produces hairpin dsRNA through transcription. Moreover, we show that these RQC factors are nuclear and cytoplasmic and are found in two RNA degradation foci in the cytoplasm: siRNA-bodies and processing-bodies. We propose a model of single-stranded RNA tug-of-war between RQC and S-PTGS that ensures the correct partitioning of RNA substrates among these RNA degradation pathways.

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Figures

Figure 1.
Figure 1.
NMD, deadenylation and exosome mutants enhance transgene S-PTGS. (A and B) The percentage of silenced plants determined by GUS quantitative protein assays in the indicated mutant and control lines. The number of plants analysed is indicated above each bar. (C and D) RNA gel blot analyses of the indicated mutant and control lines. High molecular weight RNA and siRNA gel blots were hybridized with a GUS DNA probe. 25S ribosomal RNA (rRNA) and U6 small nucleolar RNA (snRNA) served as loading controls, respectively. Hc1 plants that were expressing (+) and silenced (−) for GUS were analysed. The position of GUS 24, 22 and 21 nt siRNAs is noted. Normalized values of GUS mRNA to 25S rRNA (with either Hc1 (+) or 6b4 levels set at 1.0) and GUS 24 nt and GUS 21–22 nt siRNA to U6 snRNA [with Hc1 (−) levels set at 1.0] are indicated. ND = non-detectable.
Figure 2.
Figure 2.
Expression of an artificial miRNA targeting RRP44A leads to enhanced S-PTGS. (A) RNA gel blot analyses of three different Hc1 plant lines expressing the artificial RRP44A miRNA amiR-RRP44A. Small RNA gel blots were hybridized with a GUS DNA probe or an oligonucleotide antisense to the amiR. U6 served as a loading control for small RNA. (B) Reverse transcriptase-PCR of RRP44A and RRP44B transcripts in the corresponding Hc1/amiR-RRP44A and control Hc1 seedlings. EF1alpha was used as an amplification control. Normalized values of RRP44A and RRP44B mRNA to EF1 alpha mRNA (with Hc1 levels set at 1.0) are indicated. (C) The percentage of silenced plants determined by GUS quantitative protein assays in the indicated mutant and control lines. The number of plants analysed is indicated above each bar.
Figure 3.
Figure 3.
NMD, deadenylation and exosome mutants do not impact JAP3 IR-PTGS. Eighteen-day-old control JAP3 plants and JAP3 plants containing the indicated mutations. The photo is representative of a minimum of 20 plants screened for each genotype.
Figure 4.
Figure 4.
Subcellular localization of NMD, deadenylation, exosome and PTGS components. Confocal sections and their corresponding bright-field images of N. benthamiana leaves expressing the indicated proteins fused to GFP. The arrowheads indicate cytoplasmic foci, whereas ‘n’ labels the nucleus. Scale bars are shown on the images.
Figure 5.
Figure 5.
UPF1, PARN and CCR4a associate with both P- and siRNA-bodies. (A and B) Confocal sections of N. benthamiana leaves co-expressing the indicated fluorescent fusion proteins. Co-expression of UPF1, PARN and CCR4a with DCP1, a P-bodies marker (A), or with SGS3, a siRNA-bodies marker (B). White arrowheads indicate co-localization, and open arrowheads indicate foci positive for only one of the two fusion proteins. The area enclosed in the dashed box is shown in the close-up view. (C) Percentage of UPF1, PARN and CCR4a foci that co-localize with either P-bodies (as marked by DCP1) or siRNA-bodies (as marked by SGS3). Percentage of foci co-localizing (black) or not co-localizing (grey) with DCP1 and SGS3. The total number of foci counted is indicated above each bar. DCP1 and SGS3 foci were never observed to overlap. (D) Confocal sections of N. benthamiana leaves co-expressing SGS3, DCP1 and UPF1 fluorescent fusion proteins. Upper row: UPF1 is associated with a siRNA-body that is located adjacent to a P-body. Lower row: UPF1 is associated with a P-body that is located adjacent to two siRNA-bodies. The area enclosed in the dashed box is shown in the close-up view. Scale bars are shown on the images.
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