RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties

Schiebel, Winfried; Haas, Bernd; Marinkovic, S.; Klanner, Andrea; Sänger, Heinz L.

doi:10.1016/s0021-9258(19)50279-4

Cited by 133 publications

(11 citation statements)

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“…In many eukaryotic species, normal siRNA accumulation requires an RNA-dependent RNA polymerase (RdRP). For example in plants, RdRPs are recruited to specific template RNAs and they generate long complementary RNAs (Schiebel et al, 1993 ;Tang et al, 2003 ;Curaba and Chen, 2008). The template RNA and the RdRP product are believed to hybridize, forming a long double-stranded RNA which is subsequently cleaved by Dicer nucleases into double-stranded siRNAs (reviewed in Voinnet, 2008).…”

Section: Introductionmentioning

confidence: 99%

Functional lability of RNA-dependent RNA polymerases in animals

Pinzón

Bertrand²,

Subirana³

et al. 2018

Preprint

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RNA interference (RNAi) requires RNA-dependent RNA polymerases (RdRPs) in many eukaryotes, and RNAi amplification constitutes the only known function for eukaryotic RdRPs. Yet in animals, classical model organisms can elicit RNAi without possessing RdRPs, and only nematode RNAi was shown to require RdRPs. Here we show that RdRP genes are much more common in animals than previously thought, even in insects, where they had been assumed not to exist. RdRP genes were present in the ancestors of numerous clades, and they were subsequently lost at a high frequency. In order to probe the function of RdRPs in a deuterostome (the cephalochordate Branchiostoma lanceolatum), we performed high-throughput analyses of small RNAs from various Branchiostoma developmental stages. Our results show that Branchiostoma RdRPs do not appear to participate in RNAi: we did not detect any candidate small RNA population exhibiting classical siRNA length or sequence features. Our results show that RdRPs have been independently lost in dozens of animal clades, and even in a clade where they have been conserved (cephalochordates) their function in RNAi amplification is not preserved. Such a dramatic functional variability reveals an unexpected plasticity in RNA silencing pathways. Author summaryRNA interference (RNAi) is a conserved gene regulation system in eukaryotes. In non-animal eukaryotes, it necessitates RNA-dependent RNA polymerases ("RdRPs"). Among animals, only nematodes appear to require RdRPs for RNAi. Yet additional animal clades have RdRPs and it is assumed that they participate in RNAi. Here, we find that RdRPs are much more common in animals than previously thought, but their genes were independently lost in many lineages. Focusing on a species with RdRP genes (a cephalochordate), we found that it does not use them for RNAi. While RNAi is the only known function for eukaryotic RdRPs, our results suggest additional roles. Eukaryotic RdRPs thus have a complex evolutionary history in animals, with frequent independent losses and apparent functional diversification. November 28, 2018 1/23 Phylogenetic tree reconstruction 100 Amino acid sequences of the eukaryotic RdRP domain (Pfam #PF05183) were retrieved 101 from PFAM [35], and supplemented with the RdRP domains of the proteins identified 102 in the 538 animal proteomes (cf above). Sequences were aligned using hmmalign [36] 103 using the HMM profile of the PF05183 RdRP domain. Sequences for which the domain 104 was incomplete were deteled from the alignment. Sites used to reconstruct the 105 phylogenetic tree were selected using trimAl [37] on the Phylemon 2.0 webserver [38]. 106 Bayesian inference (BI) tree was inferred using MrBayes 3.2.6 [39], with the model 107 recommended by ProtTest 1.4 [40] under the Akaike information criterion (LG+Γ), at 108 the CIPRES Science Gateway portal [41]. Two independent runs were performed, each 109 with 4 chains and one million generations. A burn-in of 25% was used and a fifty 110 majority-rule consensus tree was calculated for the remaining ...

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Section: Introductionmentioning

confidence: 99%

Functional lability of RNA-dependent RNA polymerases in animals

Pinzón

Bertrand²,

Subirana³

et al. 2018

Preprint

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“…The first RdRP homolog found in plants was RdRP from tomato (Schiebel et al, 1993;Schiebel et al, 1998). Arabidopsis genome carries six RdRP genes: RDR1-RDR6 (Yu et al, 2003).…”

Section: Rdrpsmentioning

confidence: 99%

RNAi and miRNA pathways in plants I – molecular mechanisms

Svoboda¹

2020

Introduction to RNAi and miRNA Pathways

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RNA silencing denotes sequence-specific repression mediated by small RNAs. It includes post-transcriptional silencing mechanisms such as RNA interference (RNAi) and microRNA (miRNA) pathway and transcriptional silencing. RNA silencing regulates endogenous protein-coding gene expression, serves as a form of innate immunity targeting viruses, and protects genome integrity by repression of retrotransposons. This review provides overview of RNA silencing in flowering plants, from which comes the bulk of published data. Plants have arguably the most complex RNA silencing system among eukaryots because of existence of many paralogs of key protein factors, which form an intricate network of primary and secondary small RNAs, which mediate transcriptional and post-transcriptional effects. The first half of the review will provide a detailed catalogue of components of RNA silencing in plants while the second half will discuss the specific silencing mechanisms themselves and their biological roles, with a particular focus on the miRNA pathway, PTGS/VIG (plant's equivalent of RNAi), and transcriptional gene silencing (TGS), which is a unique RNA silencing adaptation existing in plants.

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“…The RDR6 is postulated to initiate transcription predominantly from the very 3ʹ of the target transcript (Schiebel et al, 1993) and not from an internal site, for example, the siRNA cleavage site (Moissiard et al, 2007). Furthermore, it has been shown that the 3ʹ cleavage fragment of the abRNA predominantly contributes to the RDR6‐mediated transitivity.…”

Section: Introductionmentioning

confidence: 99%

High‐pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing

Uslu

Dalakouras²,

Steffens

et al. 2022

The Plant Journal

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In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double-stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)-expressing Nicotiana benthamiana 16C line, high-pressure spraying protocol, and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3ʹ of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5ʹ and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siR-NAs and that dsRNA synthesis appears to be generally initiated at the 3ʹ-end of the target RNA.

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RNA-directed RNA polymerase from tomato leaves. II. Catalytic in vitro properties

Cited by 133 publications

References 0 publications

Functional lability of RNA-dependent RNA polymerases in animals

Functional lability of RNA-dependent RNA polymerases in animals

RNAi and miRNA pathways in plants I – molecular mechanisms

High‐pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing

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