Distinct substrate specificities of Arabidopsis DCL3 and DCL4

Nagano, Hideaki; Fukudome, Akihito; Hiraguri, Akihiro; Moriyama, Hiromitsu; Fukuhara, Teruyuki

doi:10.1093/nar/gkt1077

Cited by 80 publications

(100 citation statements)

References 40 publications

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“…These results support the idea that exogenously supplied HYL1 antagonizes BYL-maintained dsRNA processing activities relying on endogenous DCL3 and DCL4, while exogenous DRB4 assists the DCL4-mediated activity specifically. Similar dsRNA-processing properties have been established previously in Arabidopsis cell extracts (Nagano et al 2014).…”

Section: Resultssupporting

confidence: 82%

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing

Iki

Tschopp

Voinnet

2017

RNA

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Phytoviruses encode viral suppressors of RNA silencing (VSRs) to counteract the plant antiviral silencing response, which relies on virus-derived small interfering (si)RNAs processed by Dicer RNaseIII enzymes and subsequently loaded into ARGONAUTE (AGO) effector proteins. Here, a tobacco cell-free system was engineered to recapitulate the key steps of antiviral RNA silencing and, in particular, the most upstream double-stranded (ds)RNA processing reaction, not kinetically investigated thus far in the context of plant VSR studies. Comparative biochemical analyses of distinct VSRs in the reconstituted assay showed that in all cases tested, VSR interactions with siRNA duplexes inhibited the loading, but not the activity, of antiviral AGO1 and AGO2. Turnip crinkle virus P38 displayed the additional and unique property to bind both synthetic and RNA-dependent-RNA-polymerase-generated long dsRNAs, and inhibited the processing into siRNAs. Single amino acid substitutions in P38 could dissociate dsRNAprocessing from AGO-loading inhibition in vitro and in vivo, illustrating dual-inhibitory strategies discriminatively deployed within a single viral protein, which, we further show, are bona fide suppressor functions that evolved independently of the conserved coat protein function of P38.

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Section: Resultssupporting

confidence: 82%

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing

Iki

Tschopp

Voinnet

2017

RNA

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“…S1a). The short length of P4RNAs fits well with the specific preference of DCL3 for short (30 to 50 base pairs, bp) dsRNA substrates (Nagano et al, 2014), consistent with the hypothesis that these short P4RNAs serve as the precursors for siRNA biogenesis.…”

Section: Resultssupporting

confidence: 81%

A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis

Zhai

Bischof

Wang

et al. 2015

Cell

252

319

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SUMMARY RNA-directed DNA methylation in Arabidopsis thaliana is driven by the plant-specific RNA Polymerase IV (Pol IV). It has been assumed that a Pol IV transcript can give rise to multiple 24-nucleotide (nt) small interfering RNAs (siRNAs) that target DNA methylation. Here, we demonstrate that Pol IV-dependent RNAs (P4RNAs) from wild-type Arabidopsis are surprisingly short in length (30-to-40 nt) and mirror 24-nt siRNAs in distribution, abundance, strand bias, and 5’-adenine preference. P4RNAs exhibit transcription-start-sites similar to Pol II products, and are featured with 5’-monophosphates and 3’-misincorporated nucleotides. The 3’-misincorporation preferentially occurs at methylated cytosines on the template DNA strand, suggesting a co-transcriptional feedback to siRNA biogenesis by DNA methylation to reinforce silencing locally. These results highlight an unusual mechanism of Pol IV transcription and suggest a “one-precursor, one-siRNA” model for the biogenesis of 24-nt siRNAs in Arabidopsis.

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“…Interestingly, biochemical characterization of Arabidopsis DCL3 and DCL4 revealed that DCL3 preferentially cleaves short dsRNAs (37 to 50 nucleotides), whereas DCL4 preferentially cleaves long dsRNAs (Nagano et al, 2014). This result is consistent with DCL4 producing phased 21-nucleotide siRNAs over a long distance on TAS genes (Vazquez et al, 2004;Allen et al, 2005), whereas DCL3 does not.…”

Section: Discussionmentioning

confidence: 56%

Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation

Elvira-Matelot

Hachet²,

Shamandi

et al. 2016

Plant Cell

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(J.S.-V.) RNaseIII enzymes catalyze the cleavage of double-stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis thaliana RNASE THREE LIKE2 (RTL2), which carries one RNaseIII and two dsRNA binding (DRB) domains, is a unique Arabidopsis RNaseIII enzyme resembling the budding yeast small interfering RNA (siRNA)-producing Dcr1 enzyme. Here, we show that RTL2 modulates the production of a subset of small RNAs and that this activity depends on both its RNaseIII and DRB domains. However, the mode of action of RTL2 differs from that of Dcr1. Whereas Dcr1 directly cleaves dsRNAs into 23-nucleotide siRNAs, RTL2 likely cleaves dsRNAs into longer molecules, which are subsequently processed into small RNAs by the DICER-LIKE enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2-dependent loci) or reduces (RTL2-sensitive loci) the production of small RNAs. Because the vast majority of RTL2-regulated loci correspond to transposons and intergenic regions producing 24-nucleotide siRNAs that guide DNA methylation, RTL2 depletion modifies DNA methylation in these regions. Nevertheless, 13% of RTL2-regulated loci correspond to protein-coding genes. We show that changes in 24-nucleotide siRNA levels also affect DNA methylation levels at such loci and inversely correlate with mRNA steady state levels, thus implicating RTL2 in the regulation of proteincoding gene expression.

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Distinct substrate specificities of Arabidopsis DCL3 and DCL4

Cited by 80 publications

References 40 publications

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing

Biochemical and genetic functional dissection of the P38 viral suppressor of RNA silencing

A One Precursor One siRNA Model for Pol IV-Dependent siRNA Biogenesis

Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation

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