Unknown Areas of Activity of Human Ribonuclease Dicer: A Putative Deoxyribonuclease Activity

The Dicer ribonuclease plays a crucial role in the biogenesis of small regulatory RNAs (srRNAs) by processing long double-stranded RNAs and single-stranded hairpin RNA precursors into small interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Dicer-generated srRNAs can control gene expression by targeting complementary transcripts and repressing their translation or inducing their cleavage. Human Dicer (hDicer) is a multidomain enzyme comprising a putative helicase domain, a DUF283 domain, platform, a PAZ domain, a connector helix, two RNase III domains (RNase IIIa and RNase IIIb) and a dsRNA-binding domain. Specific, ~20-base pair siRNA or miRNA duplexes with 2 nucleotide (nt) 3’-overhangs are generated by Dicer when an RNA substrate is anchored within the platform-PAZ-connector helix (PPC) region. However, increasing number of reports indicate that in the absence of the PAZ domain, binding of RNA substrates can occur by other Dicer domains. Interestingly, truncated variants of Dicer, lacking the PPC region, have been found to display a DNase activity. Inspired by these findings, we investigated how the lack of the PAZ domain, or the entire PPC region, would influence the cleavage activity of hDicer. Using immunopurified 3xFlag-hDicer produced in human cells and its two variants: one lacking the PAZ domain, and the other lacking the entire PPC region, we show that the PAZ domain deletion variants of hDicer are not able to process a pre-miRNA substrate, a dsRNA with 2-nt 3ʹ-overhangs, and a blunt-ended dsRNA. However, the PAZ deletion variants exhibit both RNase and DNase activity on short single-stranded RNA and DNAs, respectively. Collectively, our results indicate that when the PAZ domain is absent, other hDicer domains may contribute to substrate binding and in this case, non-canonical products can be generated.

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Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

PLoS Pathogens ◽

10.1371/journal.ppat.1010202 ◽

2022 ◽

Vol 18 (1) ◽

pp. e1010202

Author(s):

Rommel J. Gestuveo ◽

Rhys Parry ◽

Laura B. Dickson ◽

Sebastian Lequime ◽

Vattipally B. Sreenu ◽

...

Keyword(s):

Amino Acids ◽

Antiviral Activity ◽

Aedes Aegypti ◽

Semliki Forest Virus ◽

Mutational Analysis ◽

Helicase Domain ◽

Small Interfering Rnas ◽

Rnase Iii ◽

Argonaute 2 ◽

Sirna Pathway

The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3’ overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.

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Genetic Insight into the Domain Structure and Functions of Dicer-Type Ribonucleases

International Journal of Molecular Sciences ◽

10.3390/ijms22020616 ◽

2021 ◽

Vol 22 (2) ◽

pp. 616

Author(s):

Kinga Ciechanowska ◽

Maria Pokornowska ◽

Anna Kurzyńska-Kokorniak

Keyword(s):

Rna Binding ◽

Sequence Similarity ◽

Helicase Domain ◽

Rnase Iii ◽

High Sequence Similarity ◽

Small Regulatory Rnas ◽

Domain Of Unknown Function ◽

Invertebrate Animals ◽

The Individual ◽

Insight Into

Ribonuclease Dicer belongs to the family of RNase III endoribonucleases, the enzymes that specifically hydrolyze phosphodiester bonds found in double-stranded regions of RNAs. Dicer enzymes are mostly known for their essential role in the biogenesis of small regulatory RNAs. A typical Dicer-type RNase consists of a helicase domain, a domain of unknown function (DUF283), a PAZ (Piwi-Argonaute-Zwille) domain, two RNase III domains, and a double-stranded RNA binding domain; however, the domain composition of Dicers varies among species. Dicer and its homologues developed only in eukaryotes; nevertheless, the two enzymatic domains of Dicer, helicase and RNase III, display high sequence similarity to their prokaryotic orthologs. Evolutionary studies indicate that a combination of the helicase and RNase III domains in a single protein is a eukaryotic signature and is supposed to be one of the critical events that triggered the consolidation of the eukaryotic RNA interference. In this review, we provide the genetic insight into the domain organization and structure of Dicer proteins found in vertebrate and invertebrate animals, plants and fungi. We also discuss, in the context of the individual domains, domain deletion variants and partner proteins, a variety of Dicers’ functions not only related to small RNA biogenesis pathways.

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Phytophthora effector targets a novel component of small RNA pathway in plants to promote infection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1421475112 ◽

2015 ◽

Vol 112 (18) ◽

pp. 5850-5855 ◽

Cited By ~ 82

Author(s):

Yongli Qiao ◽

Jinxia Shi ◽

Yi Zhai ◽

Yingnan Hou ◽

Wenbo Ma

Keyword(s):

Small Rna ◽

Rna Silencing ◽

Effector Proteins ◽

Helicase Domain ◽

Small Interfering Rnas ◽

Developmental Defects ◽

Interacting Protein ◽

Homologous Genes ◽

Unidentified Component ◽

Virulence Target

A broad range of parasites rely on the functions of effector proteins to subvert host immune response and facilitate disease development. The notorious Phytophthora pathogens evolved effectors with RNA silencing suppression activity to promote infection in plant hosts. Here we report that the Phytophthora Suppressor of RNA Silencing 1 (PSR1) can bind to an evolutionarily conserved nuclear protein containing the aspartate–glutamate–alanine–histidine-box RNA helicase domain in plants. This protein, designated PSR1-Interacting Protein 1 (PINP1), regulates the accumulation of both microRNAs and endogenous small interfering RNAs in Arabidopsis. A null mutation of PINP1 causes embryonic lethality, and silencing of PINP1 leads to developmental defects and hypersusceptibility to Phytophthora infection. These phenotypes are reminiscent of transgenic plants expressing PSR1, supporting PINP1 as a direct virulence target of PSR1. We further demonstrate that the localization of the Dicer-like 1 protein complex is impaired in the nucleus of PINP1-silenced or PSR1-expressing cells, indicating that PINP1 may facilitate small RNA processing by affecting the assembly of dicing complexes. A similar function of PINP1 homologous genes in development and immunity was also observed in Nicotiana benthamiana. These findings highlight PINP1 as a previously unidentified component of RNA silencing that regulates distinct classes of small RNAs in plants. Importantly, Phytophthora has evolved effectors to target PINP1 in order to promote infection.

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The conserved single-cleavage mechanism of animal DROSHA enzymes

Communications Biology ◽

10.1038/s42003-021-02860-1 ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Thuy Linh Nguyen ◽

Trung Duc Nguyen ◽

Tuan Anh Nguyen

Keyword(s):

The Other ◽

Cellular Functions ◽

Rnase Iii ◽

Internal Loop ◽

Cleavage Activity ◽

Dsrna Binding ◽

Human Rnase ◽

The One ◽

Enzymatic Mechanisms ◽

Lower Stem

AbstractRNase III enzymes typically cleave both strands of double-stranded RNAs (dsRNAs). We recently discovered that a human RNase III, DROSHA, exhibits a single cleavage on the one strand of primary microRNAs (pri-miRNAs). This study revealed that DROSHAs from the other animals, including worms and flies, also show the single cleavage on dsRNAs. Furthermore, we demonstrated that the mechanism of single cleavage is conserved in animal DROSHA enzymes. In addition, the dsRNA-binding domain (dsRBD) and a 3p-strand cleavage-supporting helix (3pCSH) of the DROSHA enzymes foster a weak single cleavage on one strand, which ensures their double cleavages. Disrupting the interaction of dsRBD-RNA and 3pCSH-RNA by an internal loop (IL) and a 3pCSH-loop in the lower stem of pri-miRNAs, respectively, inhibits one of the double cleavages of DROSHAs, and this results in the single cleavage. Our findings expand our understanding of the enzymatic mechanisms of animal DROSHAs. They also indicate that there are currently unknown cellular functions of DROSHA enzymes using their single cleavage activity.

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Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems

Development ◽

10.1242/dev.126.23.5231 ◽

1999 ◽

Vol 126 (23) ◽

pp. 5231-5243 ◽

Cited By ~ 1

Author(s):

S.E. Jacobsen ◽

M.P. Running ◽

E.M. Meyerowitz

Keyword(s):

Cell Division ◽

Rna Helicase ◽

Helicase Domain ◽

Rnase Iii ◽

Dead Box ◽

Processing Event ◽

Recessive Mutant ◽

Floral Meristems ◽

Axillary Inflorescence

Arabidopsis thaliana floral meristems are determinate structures that produce a defined number of organs, after which cell division ceases. A new recessive mutant, carpel factory (caf), converts the floral meristems to an indeterminate state. They produce extra whorls of stamens, and an indefinite number of carpels. Thus, CAF appears to suppress cell division in floral meristems. The function of CAF is partially redundant with the function of the CLAVATA (CLV) and SUPERMAN (SUP) genes, as caf clv and caf sup double mutants show dramatically enhanced floral meristem over-proliferation. caf mutant plants also show other defects, including absence of axillary inflorescence meristems, and abnormally shaped leaves and floral organs. The CAF gene was cloned and found to encode a putative protein of 1909 amino acids containing an N-terminal DExH/DEAD-box type RNA helicase domain attached to a C-terminal RNaseIII-like domain. A very similar protein of unknown function is encoded by a fungal and an animal genome. Helicase proteins are involved in a number of processes, including specific mRNA localization and mRNA splicing. RNase III proteins are involved in the processing of rRNA and some mRNA molecules. Thus CAF may act through some type of RNA processing event(s). CAF gives rise to two major transcripts of 2.5 and 6.2 kb. In situ hybridization experiments show that CAF RNA is expressed throughout all shoot tissues.

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The Evolutionary Significance of RNAi in the Fungal Kingdom

International Journal of Molecular Sciences ◽

10.3390/ijms21249348 ◽

2020 ◽

Vol 21 (24) ◽

pp. 9348

Author(s):

Carlos Lax ◽

Ghizlane Tahiri ◽

José Alberto Patiño-Medina ◽

José T. Cánovas-Márquez ◽

José A. Pérez-Ruiz ◽

...

Keyword(s):

Target Genes ◽

Regulation Of Gene Expression ◽

Genome Integrity ◽

Evolutionary Significance ◽

Messenger Rnas ◽

Small Interfering Rnas ◽

Rnase Iii ◽

Double Stranded Rna ◽

Main Component ◽

Fungal Kingdom

RNA interference (RNAi) was discovered at the end of last millennium, changing the way scientists understood regulation of gene expression. Within the following two decades, a variety of different RNAi mechanisms were found in eukaryotes, reflecting the evolutive diversity that RNAi entails. The essential silencing mechanism consists of an RNase III enzyme called Dicer that cleaves double-stranded RNA (dsRNA) generating small interfering RNAs (siRNAs), a hallmark of RNAi. These siRNAs are loaded into the RNA-induced silencing complex (RISC) triggering the cleavage of complementary messenger RNAs by the Argonaute protein, the main component of the complex. Consequently, the expression of target genes is silenced. This mechanism has been thoroughly studied in fungi due to their proximity to the animal phylum and the conservation of the RNAi mechanism from lower to higher eukaryotes. However, the role and even the presence of RNAi differ across the fungal kingdom, as it has evolved adapting to the particularities and needs of each species. Fungi have exploited RNAi to regulate a variety of cell activities as different as defense against exogenous and potentially harmful DNA, genome integrity, development, drug tolerance, or virulence. This pathway has offered versatility to fungi through evolution, favoring the enormous diversity this kingdom comprises.

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Binding and processing of small dsRNA molecules by the class 1 RNase III protein encoded by sweet potato chlorotic stunt virus

Journal of General Virology ◽

10.1099/vir.0.058693-0 ◽

2014 ◽

Vol 95 (2) ◽

pp. 486-495 ◽

Cited By ~ 8

Author(s):

Isabel Weinheimer ◽

Kajohn Boonrod ◽

Mirko Moser ◽

Michael Wassenegger ◽

Gabi Krczal ◽

...

Keyword(s):

Sweet Potato ◽

Transcriptional Gene Silencing ◽

Specific Class ◽

Dependent Manner ◽

Small Interfering Rnas ◽

Rnase Iii ◽

Potato Plants ◽

Post Transcriptional Gene Silencing ◽

Class 1

Sweet potato chlorotic stunt virus (SPCSV; genus Crinivirus, family Closteroviridae) causes heavy yield losses in sweet potato plants co-infected with other viruses. The dsRNA-specific class 1 RNase III–like endoribonuclease (RNase3) encoded by SPCSV suppresses post-transcriptional gene silencing and eliminates antiviral defence in sweet potato plants in an endoribonuclease activity-dependent manner. RNase3 can cleave long dsRNA molecules, synthetic small interfering RNAs (siRNAs), and plant- and virus-derived siRNAs extracted from sweet potato plants. In this study, conditions for efficient expression and purification of enzymically active recombinant RNase3 were established. Similar to bacterial class 1 RNase III enzymes, RNase3-Ala (a dsRNA cleavage-deficient mutant) bound to and processed double-stranded siRNA (ds-siRNA) as a dimer. The results support the classification of SPCSV RNase3 as a class 1 RNase III enzyme. There is little information about the specificity of RNase III enzymes on small dsRNAs. In vitro assays indicated that ds-siRNAs and microRNAs (miRNAs) with a regular A-form conformation were cleaved by RNase3, but asymmetrical bulges, extensive mismatches and 2′-O-methylation of ds-siRNA and miRNA interfered with processing. Whereas Mg2+ was the cation that best supported the catalytic activity of RNase3, binding of 21 nt small dsRNA molecules was most efficient in the presence of Mn2+. Processing of long dsRNA by RNase3 was efficient at pH 7.5 and 8.5, whereas ds-siRNA was processed more efficiently at pH 8.5. The results revealed factors that influence binding and processing of small dsRNA substrates by class 1 RNase III in vitro or make them unsuitable for processing by the enzyme.

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MicroRNA-independent roles of the RNase III enzymes Drosha and Dicer

Open Biology ◽

10.1098/rsob.130144 ◽

2013 ◽

Vol 3 (10) ◽

pp. 130144 ◽

Cited By ~ 48

Author(s):

Timothy M. Johanson ◽

Andrew M. Lew ◽

Mark M. W. Chong

Keyword(s):

Ribosomal Rna ◽

Messenger Rnas ◽

Small Interfering Rnas ◽

Rnase Iii ◽

Ribonuclease Iii ◽

Stem Loop ◽

Viral Rnas ◽

Genome Regulation ◽

Many Sources ◽

Stem Cell Populations

The ribonuclease III enzymes Drosha and Dicer are renowned for their central roles in the biogenesis of microRNAs (miRNAs). For many years, this has overshadowed the true versatility and importance of these enzymes in the processing of other RNA substrates. For example, Drosha also recognizes and cleaves messenger RNAs (mRNAs), and potentially ribosomal RNA. The cleavage of mRNAs occurs via recognition of secondary stem-loop structures similar to miRNA precursors, and is an important mechanism of repressing gene expression, particularly in progenitor/stem cell populations. On the other hand, Dicer also has critical roles in genome regulation and surveillance. These include the production of endogenous small interfering RNAs from many sources, and the degradation of potentially harmful short interspersed element and viral RNAs. These findings have sparked a renewed interest in these enzymes, and their diverse functions in biology.

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Loquacious-PD facilitatesDrosophilaDicer-2 cleavage through interactions with the helicase domain and dsRNA

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707063114 ◽

2017 ◽

Vol 114 (38) ◽

pp. E7939-E7948 ◽

Cited By ~ 8

Author(s):

Kyle D. Trettin ◽

Niladri K. Sinha ◽

Debra M. Eckert ◽

Sarah E. Apple ◽

Brenda L. Bass

Keyword(s):

Amino Acids ◽

Binding Motif ◽

Helicase Domain ◽

C Terminus ◽

Dsrna Binding ◽

Interaction Interface ◽

Mechanistic Basis ◽

Dsrna Cleavage

Loquacious-PD (Loqs-PD) is required for biogenesis of many endogenous siRNAs inDrosophila. In vitro, Loqs-PD enhances the rate of dsRNA cleavage by Dicer-2 and also enables processing of substrates normally refractory to cleavage. Using purified components, and Loqs-PD truncations, we provide a mechanistic basis for Loqs-PD functions. Our studies indicate that the 22 amino acids at the C terminus of Loqs-PD, including an FDF-like motif, directly interact with the Hel2 subdomain of Dicer-2’s helicase domain. This interaction is RNA-independent, but we find that modulation of Dicer-2 cleavage also requires dsRNA binding by Loqs-PD. Furthermore, while the first dsRNA-binding motif of Loqs-PD is dispensable for enhancing cleavage of optimal substrates, it is essential for enhancing cleavage of suboptimal substrates. Finally, our studies define a previously unrecognized Dicer interaction interface and suggest that Loqs-PD is well positioned to recruit substrates into the helicase domain of Dicer-2.

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The N-Terminal Domain That Distinguishes Yeast from Bacterial RNase III Contains a Dimerization Signal Required for Efficient Double-Stranded RNA Cleavage

Molecular and Cellular Biology ◽

10.1128/mcb.20.4.1104-1115.2000 ◽

2000 ◽

Vol 20 (4) ◽

pp. 1104-1115 ◽

Cited By ~ 38

Author(s):

Bruno Lamontagne ◽

Annie Tremblay ◽

Sherif Abou Elela

Keyword(s):

Size Exclusion ◽

Rna Cleavage ◽

Rnase Iii ◽

Double Stranded Rna ◽

Terminal Domain ◽

Dsrna Binding ◽

Exclusion Chromatography ◽

Dsrna Cleavage ◽

Two Hybrid

ABSTRACT Yeast Rnt1 is a member of the double-stranded RNA (dsRNA)-specific RNase III family identified by conserved dsRNA binding (dsRBD) and nuclease domains. Comparative sequence analyses have revealed an additional N-terminal domain unique to the eukaryotic homologues of RNase III. The deletion of this domain from Rnt1 slowed growth and led to mild accumulation of unprocessed 25S pre-rRNA. In vitro, deletion of the N-terminal domain reduced the rate of RNA cleavage under physiological salt concentration. Size exclusion chromatography and cross-linking assays indicated that the N-terminal domain and the dsRBD self-interact to stabilize the Rnt1 homodimer. In addition, an interaction between the N-terminal domain and the dsRBD was identified by a two-hybrid assay. The results suggest that the eukaryotic N-terminal domain of Rnt1 ensures efficient dsRNA cleavage by mediating the assembly of optimum Rnt1-RNA ribonucleoprotein complex.

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