scholarly journals Functional specialization of monocot DCL3 and DCL5 proteins through the evolution of the PAZ domain

2021 ◽  
Author(s):  
Shirui Chen ◽  
Wei Liu ◽  
Masahiro Naganuma ◽  
Yukihide Tomari ◽  
Hiro-oki Iwakawa
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Functional Differentiation ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Secondary Sirnas ◽  
Fixed End ◽  
Paz Domain ◽  
Reproductive Processes

Monocot DICER-LIKE3 (DCL3) and DCL5 produce distinct 24-nt heterochromatic small interfering RNAs (hc-siRNAs) and phased secondary siRNAs (phasiRNAs). The former small RNAs are linked to plant heterochromatin, and the latter to reproductive processes. It is assumed that these DCLs evolved from an ancient "eudicot-type" DCL3 ancestor, which may have produced both types of siRNAs. However, how functional differentiation was achieved after gene duplication remains elusive. Here, we find that monocot DCL3 and DCL5 exhibit biochemically distinct preferences for 3′ overhangs and 5′ phosphates, consistent with the structural properties of their in vivo double-stranded RNA substrates. Importantly, these distinct substrate specificities are determined by the PAZ domains of DCL3 and DCL5 which have accumulated mutations during the course of evolution. These data explain the mechanism by which these DCLs cleave their cognate substrates from a fixed end, ensuring the production of functional siRNAs. Our study also indicates how plants have diversified and optimized RNA silencing mechanisms during evolution.

scholarly journals Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production

2016 ◽  
Vol 113 (49) ◽  
pp. 14031-14036 ◽  
Author(s):  
Suresh K. Kandasamy ◽  
Ryuya Fukunaga
Keyword(s):  
Rna Silencing ◽  
Point Mutations ◽  
Binding Pocket ◽  
Rna Cleavage ◽  
High Fidelity ◽  
Small Interfering Rnas ◽  
Phosphate Binding ◽  
Micro Rnas ◽  
Paz Domain

The enzyme Dicer produces small silencing RNAs such as micro-RNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22–24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5′-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5′-monophosphate of a long dsRNA substrate is anchored by the phosphate-binding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.

scholarly journals Diverging Affinity of Tospovirus RNA Silencing Suppressor Proteins, NSs, for Various RNA Duplex Molecules

2010 ◽  
Vol 84 (21) ◽  
pp. 11542-11554 ◽  
Author(s):  
Esther Schnettler ◽  
Hans Hemmes ◽  
Rik Huismann ◽  
Rob Goldbach ◽  
Marcel Prins ◽  
...  
Keyword(s):  
Rna Silencing ◽  
Fluorescent Protein ◽  
Micro Rna ◽  
Impatiens Necrotic Spot Virus ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Cell Extracts ◽  
Green Fluorescent

ABSTRACT The tospovirus NSs protein was previously shown to suppress the antiviral RNA silencing mechanism in plants. Here the biochemical analysis of NSs proteins from different tospoviruses, using purified NSs or NSs containing cell extracts, is described. The results showed that all tospoviral NSs proteins analyzed exhibited affinity to small double-stranded RNA molecules, i.e., small interfering RNAs (siRNAs) and micro-RNA (miRNA)/miRNA* duplexes. Interestingly, the NSs proteins from tomato spotted wilt virus (TSWV), impatiens necrotic spot virus (INSV), and groundnut ringspot virus (GRSV) also showed affinity to long double-stranded RNA (dsRNA), whereas tomato yellow ring virus (TYRV) NSs did not. The TSWV NSs protein was shown to be capable of inhibiting Dicer-mediated cleavage of long dsRNA in vitro. In addition, it suppressed the accumulation of green fluorescent protein (GFP)-specific siRNAs during coinfiltration with an inverted-repeat-GFP RNA construct in Nicotiana benthamiana. In vivo interference of TSWV NSs in the miRNA pathway was shown by suppression of an enhanced GFP (eGFP) miRNA sensor construct. The ability to stabilize miRNA/miRNA* by different tospovirus NSs proteins in vivo was demonstrated by increased accumulation and detection of both miRNA171c and miRNA171c* in tospovirus-infected N. benthamiana. All together, these data suggest that tospoviruses interfere in the RNA silencing pathway by sequestering siRNA and miRNA/miRNA* molecules before they are uploaded into their respective RNA-induced silencing complexes. The observed affinity to long dsRNA for only a subset of the tospoviruses studied is discussed in light of evolutional divergence and their ancestral relation to the animal-infecting members of the Bunyaviridae.

RNA-dependent RNA polymerases in RNA silencing

2011 ◽  
Vol 392 (4) ◽  
Author(s):  
Yoshiko Maida ◽  
Kenkichi Masutomi
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Rna Polymerases ◽  
Telomerase Reverse Transcriptase ◽  
Structural Homology ◽  
Small Interfering Rnas ◽  
Human Telomerase Reverse Transcriptase ◽  
Human Telomerase ◽  
Traditional Approaches

Abstract RNA-dependent RNA polymerases (RdRPs) synthesize double-stranded RNAs that are processed into small RNAs and mediate gene silencing. Viral RdRPs and cellular RdRPs show little structural homology to each other. Cellular RdRPs play key roles in RNA silencing by producing complementary strands for target RNAs via Dicer-dependent and -independent mechanisms. Although the existence of a functional mammalian homolog of RdRP has long been predicted, traditional approaches to identify such enzymes were unsuccessful. Recently, human telomerase reverse transcriptase, a polymerase closely related to viral RdRPs, has been shown to function as an RdRP and contributes to RNA silencing in vivo. These findings suggest that endogenous small interfering RNAs are produced by several mechanisms in eukaryotes.

Viroid-induced DNA methylation in plants

2013 ◽  
Vol 4 (6) ◽  
pp. 557-565 ◽  
Author(s):  
Athanasios Dalakouras ◽  
Elena Dadami ◽  
Michael Wassenegger
Keyword(s):  
Dna Methylation ◽  
Small Rnas ◽  
Rna Silencing ◽  
De Novo ◽  
Rna Degradation ◽  
Double Stranded Rna ◽  
Methyl Group ◽  
Rna Molecules ◽  
Cumulative Data

AbstractIn eukaryotes, DNA methylation refers to the addition of a methyl group to the fifth atom in the six-atom ring of cytosine residues. At least in plants, DNA regions that become de novo methylated can be defined by homologous RNA molecules in a process termed RNA-directed DNA methylation (RdDM). RdDM was first discovered in viroid-infected plants. Viroids are pathogenic circular, non-coding, single-stranded RNA molecules. Members of the Pospiviroidae family replicate in the nucleus through double-stranded RNA intermediates, attracting the host RNA silencing machinery. The recruitment of this machinery results in the production of viroid-derived small RNAs (vd-sRNAs) that mediate RNA degradation and DNA methylation of cognate sequences. Here, we provide an overview of the cumulative data on the field of viroid-induced RdDM and discuss three possible scenarios concerning the mechanistic details of its establishment.

scholarly journals Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

2020 ◽  
Vol 48 (7) ◽  
pp. 3906-3921 ◽  
Author(s):  
Volker Nitschko ◽  
Stefan Kunzelmann ◽  
Thomas Fröhlich ◽  
Georg J Arnold ◽  
Klaus Förstemann
Keyword(s):  
Small Rnas ◽  
Rna Binding ◽  
Small Interfering Rnas ◽  
Double Stranded Rna ◽  
Binding Domains ◽  
Convergent Transcription ◽  
Dsrna Binding ◽  
Functional Screens

Abstract RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

Distinct Populations of Primary and Secondary Effectors During RNAi in C. elegans

Science ◽  
2006 ◽  
Vol 315 (5809) ◽  
pp. 241-244 ◽  
Author(s):  
Julia Pak ◽  
Andrew Fire
Keyword(s):  
Rna Interference ◽  
Small Rnas ◽  
De Novo ◽  
Messenger Rnas ◽  
Cellular Regulation ◽  
Rdrp Activity ◽  
Antisense Transcripts ◽  
Double Stranded Rna ◽  
C Elegans ◽  
Secondary Sirnas

RNA interference (RNAi) is a phylogenetically widespread gene-silencing process triggered by double-stranded RNA. In plants and Caenorhabditis elegans, two distinct populations of small RNAs have been proposed to participate in RNAi: “Primary siRNAs” (derived from DICER nuclease-mediated cleavage of the original trigger) and “secondary siRNAs” [additional small RNAs whose synthesis requires an RNA-directed RNA polymerase (RdRP)]. Analyzing small RNAs associated with ongoing RNAi in C. elegans, we found that secondary siRNAs constitute the vast majority. The bulk of secondary siRNAs exhibited structure and sequence indicative of a biosynthetic mode whereby each molecule derives from an independent de novo initiation by RdRP. Analysis of endogenous small RNAs indicated that a fraction derive from a biosynthetic mechanism that is similar to that of secondary siRNAs formed during RNAi, suggesting that small antisense transcripts derived from cellular messenger RNAs by RdRP activity may have key roles in cellular regulation.

scholarly journals Secondary siRNAs in Plants: Biosynthesis, Various Functions, and Applications in Virology

2021 ◽  
Vol 12 ◽  
Author(s):  
Neeti Sanan-Mishra ◽  
A. Abdul Kader Jailani ◽  
Bikash Mandal ◽  
Sunil K. Mukherjee
Keyword(s):  
Gene Silencing ◽  
Small Rnas ◽  
Rna Silencing ◽  
Viral Infections ◽  
Natural Resistance ◽  
Virus Induced Gene Silencing ◽  
Systemic Spread ◽  
Secondary Sirnas ◽  
Silencing Pathways ◽  
Virus Interactions

The major components of RNA silencing include both transitive and systemic small RNAs, which are technically called secondary sRNAs. Double-stranded RNAs trigger systemic silencing pathways to negatively regulate gene expression. The secondary siRNAs generated as a result of transitive silencing also play a substantial role in gene silencing especially in antiviral defense. In this review, we first describe the discovery and pathways of transitivity with emphasis on RNA-dependent RNA polymerases followed by description on the short range and systemic spread of silencing. We also provide an in-depth view on the various size classes of secondary siRNAs and their different roles in RNA silencing including their categorization based on their biogenesis. The other regulatory roles of secondary siRNAs in transgene silencing, virus-induced gene silencing, transitivity, and trans-species transfer have also been detailed. The possible implications and applications of systemic silencing and the different gene silencing tools developed are also described. The details on mobility and roles of secondary siRNAs derived from viral genome in plant defense against the respective viruses are presented. This entails the description of other compatible plant–virus interactions and the corresponding small RNAs that determine recovery from disease symptoms, exclusion of viruses from shoot meristems, and natural resistance. The last section presents an overview on the usefulness of RNA silencing for management of viral infections in crop plants.

scholarly journals Effects of the Crinivirus Coat Protein–Interacting Plant Protein SAHH on Post-Transcriptional RNA Silencing and Its Suppression

2013 ◽  
Vol 26 (9) ◽  
pp. 1004-1015 ◽  
Author(s):  
M. Carmen Cañizares ◽  
Rosa Lozano-Durán ◽  
Tomás Canto ◽  
Eduardo R. Bejarano ◽  
David M. Bisaro ◽  
...  
Keyword(s):  
Coat Protein ◽  
Rna Silencing ◽  
Rna Degradation ◽  
Plant Viruses ◽  
Plant Protein ◽  
Transcriptional Gene Silencing ◽  
Small Interfering Rnas ◽  
Post Transcriptional Gene Silencing ◽  
Secondary Sirnas ◽  
Tomato Chlorosis Virus

In plants, post-transcriptional gene silencing (PTGS) is a sequence-specific mechanism of RNA degradation induced by double-stranded RNA (dsRNA), which is processed into small interfering RNAs (siRNAs). siRNAs are methylated and, thereby, stabilized by the activity of the S-adenosylmethionine-dependent RNA methyltransferase HEN1. PTGS is amplified by host-encoded RNA-dependent RNA polymerases (RDR), which generate dsRNA that is processed into secondary siRNAs. To counteract this RNA silencing-mediated response of the host, plant viruses express proteins with silencing suppression activity. Here, we report that the coat protein (CP) of crinivirus (family Closteroviridae, genus Crinivirus) Tomato chlorosis virus, a known suppressor of silencing, interacts with S-adenosylhomocysteine hydrolase (SAHH), a plant protein essential for sustaining the methyl cycle and S-adenosylmethionine-dependent methyltransferase activity. Our results show that, by contributing to an increased accumulation of secondary siRNAs generated by the action of RDR6, SAHH enhances local RNA silencing. Although downregulation of SAHH prevents local silencing, it enhances the spread of systemic silencing. Our results also show that SAHH is important in the suppression of local RNA silencing not only by the crinivirus Tomato chlorosis virus CP but also by the multifunctional helper component-proteinase of the potyvirus Potato virus Y.

scholarly journals Gene Regulation Mediated by microRNA-Triggered Secondary Small RNAs in Plants

Plants ◽  
2019 ◽  
Vol 8 (5) ◽  
pp. 112 ◽  
Author(s):  
Felipe Fenselau de Felippes
Keyword(s):  
Gene Regulation ◽  
Small Rnas ◽  
Target Genes ◽  
Regulation Of Gene Expression ◽  
Small Interfering Rnas ◽  
Expression Of Genes ◽  
Secondary Sirnas ◽  
In Trans ◽  
Production Pattern ◽  
In Cis

In plants, proper development and response to abiotic and biotic stimuli requires an orchestrated regulation of gene expression. Small RNAs (sRNAs) are key molecules involved in this process, leading to downregulation of their target genes. Two main classes of sRNAs exist, the small interfering RNAs (siRNAs) and microRNAs (miRNAs). The role of the latter class in plant development and physiology is well known, with many examples of how miRNAs directly impact the expression of genes in cells where they are produced, with dramatic consequences to the life of the plant. However, there is an aspect of miRNA biology that is still poorly understood. In some cases, miRNA targeting can lead to the production of secondary siRNAs from its target. These siRNAs, which display a characteristic phased production pattern, can act in cis, reinforcing the initial silencing signal set by the triggering miRNA, or in trans, affecting genes that are unrelated to the initial target. In this review, the mechanisms and implications of this process in the gene regulation mediated by miRNAs will be discussed. This work will also explore techniques for gene silencing in plants that are based on this unique pathway.

scholarly journals Modification of Small RNAs Associated with Suppression of RNA Silencing by Tobamovirus Replicase Protein

2007 ◽  
Vol 81 (19) ◽  
pp. 10379-10388 ◽  
Author(s):  
Hannes Vogler ◽  
Rashid Akbergenov ◽  
Padubidri V. Shivaprasad ◽  
Vy Dang ◽  
Monika Fasler ◽  
...  
Keyword(s):  
Small Rnas ◽  
Rna Silencing ◽  
Fluorescent Protein ◽  
Plant Viruses ◽  
Micro Rna ◽  
Silencing Suppressor ◽  
Small Interfering Rnas ◽  
Suppressor Activity ◽  
Virus Family ◽  
Green Fluorescent

ABSTRACT Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

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