A universal small molecule,inorganic phosphate,restricts the substrate specificity of Dicer-2 in small RNA biogenesis

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme.     

The inside-out mechanism of Dicers from budding yeasts

The Dicer ribonuclease III (RNase III) enzymes process long double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that direct RNA interference. Here, we describe the structure and activity of a catalytically active fragment of Kluyveromyces polysporus Dcr1, which represents the noncanonical Dicers found in budding yeasts. The crystal structure revealed a homodimer resembling that of bacterial RNase III but extended by a unique N-terminal domain, and it identified additional catalytic residues conserved throughout eukaryotic RNase III enzymes. Biochemical analyses showed that Dcr1 dimers bind cooperatively along the dsRNA substrate such that the distance between consecutive active sites determines the length of the siRNA products. Thus, unlike canonical Dicers, which successively remove siRNA duplexes from the dsRNA termini, budding-yeast Dicers initiate processing in the interior and work outward. The distinct mechanism of budding-yeast Dicers establishes a paradigm for natural molecular rulers and imparts substrate preferences with ramifications for biological function.     

Homodimeric structure and double-stranded RNA cleavage activity of the C-terminal RNase III domain of human dicer

Human Dicer contains two RNase III domains (RNase IIIa and RNase IIIb) that are responsible for the production of short interfering RNAs and microRNAs. These small RNAs induce gene silencing known as RNA interference. Here, we report the crystal structure of the C-terminal RNase III domain (RNase IIIb) of human Dicer at 2.0 Å resolution. The structure revealed that the RNase IIIb domain can form a tightly associated homodimer, which is similar to the dimers of the bacterial RNase III domains and the two RNase III domains of Giardia Dicer. Biochemical analysis showed that the RNase IIIb homodimer can cleave double-stranded RNAs (dsRNAs), and generate short dsRNAs with 2 nt 3′ overhang, which is characteristic of RNase III products. The RNase IIIb domain contained two magnesium ions per monomer around the active site. The distance between two Mg-1 ions is approximately 20.6 Å, almost identical with those observed in bacterial RNase III enzymes and Giardia Dicer, while the locations of two Mg-2 ions were not conserved at all. We presume that Mg-1 ions act as catalysts for dsRNA cleavage, while Mg-2 ions are involved in RNA binding.     

Nucleotide sequence of miRNA precursor contributes to cleavage site selection by Dicer

The ribonuclease Dicer excises mature miRNAs from a diverse group of precursors (pre-miRNAs), most of which contain various secondary structure motifs in their hairpin stem. In this study, we analyzed Dicer cleavage in hairpin substrates deprived of such motifs. We searched for the factors other than the secondary structure, which may influence the length diversity and heterogeneity of miRNAs. We found that the nucleotide sequence at the Dicer cleavage site influences both of these miRNA characteristics. With regard to cleavage mechanism, we demonstrate that the Dicer RNase IIIA domain that cleaves within the 3′ arm of the pre-miRNA is more sensitive to the nucleotide sequence of its substrate than is the RNase IIIB domain. The RNase IIIA domain avoids releasing miRNAs with G nucleotide and prefers to generate miRNAs with a U nucleotide at the 5′ end. We also propose that the sequence restrictions at the Dicer cleavage site might be the factor that contributes to the generation of miRNA duplexes with 3′ overhangs of atypical lengths. This finding implies that the two RNase III domains forming the single processing center of Dicer may exhibit some degree of flexibility, which allows for the formation of these non-standard 3′ overhangs.     

An unusual Dicer-like1 protein fuels the RNA interference pathway in Trypanosoma brucei

RNA interference (RNAi) is an evolutionarily conserved gene-silencing pathway that is triggered by double-stranded RNA (dsRNA). Central to this pathway are two ribonucleases: Dicer, a multidomain RNase III family enzyme that initiates RNAi by generating small interfering RNAs (siRNAs), and Argonaute or Slicer, an RNase H signature enzyme that affects cleavage of mRNA. Previous studies in the early diverging protozoan Trypanosoma brucei have established a key role for Argonaute 1 in RNAi. However, the identity of Dicer has not been resolved. Here, we report the identification and functional characterization of a T. brucei Dicer-like enzyme (TbDcl1). Using genetic and biochemical approaches, we provide evidence that TbDcl1 is required for the generation of siRNA-size molecules and for RNAi. Whereas Dicer and Dicer-like proteins are endowed with two adjacent RNase III domains at the carboxyl terminus (RNase IIIa and RNase IIIb), the arrangement of these two domains is unusual in TbDcl1. RNase IIIa is close to the amino terminus, and RNase IIIb is located approximately in the center of the molecule. This domain organization is specific to trypanosomatids and further illustrates the variable structures of protozoan Dicer-like proteins as compared to fungal and metazoan Dicer.     

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

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.     

Human let-7 stem-loop precursors harbor features of RNase III cleavage products

The bidentate RNase III Dicer cleaves microRNA precursors to generate the 21–23 nt long mature RNAs. These precursors are 60–80 nt long, they fold into a characteristic stem–loop structure and they are generated by an unknown mechanism. To gain insights into the biogenesis of microRNAs, we have characterized the precise 5′ and 3′ ends of the let-7 precursors in human cells. We show that they harbor a 5′-phosphate and a 3′-OH and that, remarkably, they contain a 1–4 nt 3′ overhang. These features are characteristic of RNase III cleavage products. Since these precursors are present in both the nucleus and the cytoplasm of human cells, our results suggest that they are generated in the nucleus by the nuclear RNase III. Additionally, these precursors fit the minihelix export motif and are thus likely exported by this pathway.     

Structural and mechanistic insight into stem‐loop RNA processing by yeast Pichia stipitis Dicer

Dicer is a member of the ribonuclease III enzyme family and processes double‐stranded RNA into small functional RNAs. The variation in the domain architecture of Dicer among different species whilst preserving its biological dicing function is intriguing. Here, we describe the structure and function of a novel catalytically active RNase III protein, a non‐canonical Dicer (PsDCR1), found in budding yeast Pichia stipitis. The structure of the catalytically active region (the catalytic RNase III domain and double‐stranded RNA‐binding domain 1 [dsRBD1]) of DCR1 showed that RNaseIII domain is structurally similar to yeast RNase III (Rnt1p) but uniquely presents dsRBD1 in a diagonal orientation, forming a catalytic core made of homodimer and large RNA‐binding surface. The second dsRNA binding domain at C‐terminus, which is absent in Rnt1, enhances the RNA cleavage activity. Although the cleavage pattern of PsDCR1 anchors an apical loop similar to Rnt1, the cleavage activity depended on the sequence motif at the lower stem, not the apical loop, of hairpin RNA. Through RNA sequencing and RNA mutations, we showed that RNA cleavage by PsDCR1 is determined by the stem‐loop structure of the RNA substrate, suggesting the possibility that stem‐loop RNA‐guided gene silencing pathway exists in budding yeast.     

Ribonuclease revisited: structural insights into ribonuclease III family enzymes

Ribonuclease III (RNase III) enzymes occur ubiquitously in biology and are responsible for processing RNA precursors into functional RNAs that participate in protein synthesis, RNA interference and a range of other cellular activities. Members of the RNase III enzyme family, including Escherichia coli RNase III, Rnt1, Dicer and Drosha, share the ability to recognize and cleave double-stranded RNA (dsRNA), typically at specific positions or sequences. Recent biochemical and structural data have shed new light on how RNase III enzymes catalyze dsRNA hydrolysis and how substrate specificity is achieved. A major theme emerging from these studies is that accessory domains present in different RNase III enzymes are the key determinants of substrate selectivity, which in turn dictates the specialized biological function of each type of RNase III protein.     

Dicer结构和功能研究进展

摘要: Dicer蛋白是RNA干扰机制的关键组分, 负责siRNA和miRNA的产生。它主要由RNA解旋酶结构域、PAZ结构域、RNaseⅢ结构域和双链RNA结合结构域构成。Dicer的结构特点决定了它所产生的小RNA的结构特点。不同生物体具有不同数量的Dicer, 各Dicer既有功能上各自独立的特点, 同时又有功能的冗余和交叉, 而在进化过程中, Dicer的数量逐渐减少, 功能却逐步整合从而表现出多功能的特点。对Dicer结构和功能进行深入研究, 有助于了解Dicer乃至整个RNAi及相关途径的作用机制, 也有助于揭示它们在进化过程中所表现出的规律和特点。文章对上述Dicer结构及功能特点作简要综述。     

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is ‘sliced’ by Ago2, then 3′-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼23–26 nt products. The difference was due to the 3′ modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3′ trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3′–5′ exoribonuclease that trims ‘long’ mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.     

A ribonuclease III involved in virulence of Mucorales fungi has evolved to cut exclusively single-stranded RNA

Members of the ribonuclease III (RNase III) family regulate gene expression by processing double-stranded RNA (dsRNA). This family includes eukaryotic Dicer and Drosha enzymes that generate small dsRNAs in the RNA interference (RNAi) pathway. The fungus Mucor lusitanicus, which causes the deadly infection mucormycosis, has a complex RNAi system encompassing a non-canonical RNAi pathway (NCRIP) that regulates virulence by degrading specific mRNAs. In this pathway, Dicer function is replaced by R3B2, an atypical class I RNase III, and small single-stranded RNAs (ssRNAs) are produced instead of small dsRNA as Dicer-dependent RNAi pathways. Here, we show that R3B2 forms a homodimer that binds to ssRNA and dsRNA molecules, but exclusively cuts ssRNA, in contrast to all known RNase III. The dsRNA cleavage inability stems from its unusual RNase III domain (RIIID) because its replacement by a canonical RIIID allows dsRNA processing. A crystal structure of R3B2 RIIID resembles canonical RIIIDs, despite the low sequence conservation. However, the groove that accommodates dsRNA in canonical RNases III is narrower in the R3B2 homodimer, suggesting that this feature could be responsible for the cleavage specificity for ssRNA. Conservation of this activity in R3B2 proteins from other mucormycosis-causing Mucorales fungi indicates an early evolutionary acquisition.     

2'-O-[2-[(N,N-dimethylamino)oxy]ethyl]-modified oligonucleotides inhibit expression of mRNA in vitro and in vivo

Synthesis and antisense activity of oligonucleotides modified with 2′-O-[2-[(N,N-dimethylamino)oxy] ethyl] (2′-O-DMAOE) are described. The 2′-O-DMAOE-modified oligonucleotides showed superior metabolic stability in mice. The phosphorothioate oligonucleotide ‘gapmers’, with 2′-O-DMAOE- modified nucleoside residues at the ends and 2′-deoxy nucleosides residues in the central region, showed dose-dependent inhibition of mRNA expression in cell culture for two targets. ‘Gapmer’ oligonucleotides have one or two 2′-O-modified regions and a 2′-deoxyoligonucleotide phosphorothioate region that allows RNase H digestion of target mRNA. To determine the in vivo potency and efficacy, BalbC mice were treated with 2′-O-DMAOE gapmers and a dose-dependent reduction in the targeted C-raf mRNA expression was observed. Oligonucleotides with 2′-O-DMAOE modifications throughout the sequences reduced the intercellular adhesion molecule-1 (ICAM-1) protein expression very efficiently in HUVEC cells with an IC₅₀ of 1.8 nM. The inhibition of ICAM-1 protein expression by these uniformly modified 2′-O-DMAOE oligonucleotides may be due to selective interference with the formation of the translational initiation complex. These results demonstrate that 2′-O-DMAOE- modified oligonucleotides are useful for antisense-based therapeutics when either RNase H-dependent or RNase H-independent target reduction mechanisms are employed.     

Repeated Evolution of Inactive Pseudonucleases in a Fungal Branch of the Dis3/RNase II Family of Nucleases

The RNase II family of 3′–5′ exoribonucleases is present in all domains of life, and eukaryotic family members Dis3 and Dis3L2 play essential roles in RNA degradation. Ascomycete yeasts contain both Dis3 and inactive RNase II-like “pseudonucleases.” The latter function as RNA-binding proteins that affect cell growth, cytokinesis, and fungal pathogenicity. However, the evolutionary origins of these pseudonucleases are unknown: What sequence of events led to their novel function, and when did these events occur? Here, we show how RNase II pseudonuclease homologs, including Saccharomyces cerevisiae Ssd1, are descended from active Dis3L2 enzymes. During fungal evolution, active site mutations in Dis3L2 homologs have arisen at least four times, in some cases following gene duplication. In contrast, N-terminal cold-shock domains and regulatory features are conserved across diverse dikarya and mucoromycota, suggesting that the nonnuclease function requires these regions. In the basidiomycete pathogenic yeast Cryptococcus neoformans, the single Ssd1/Dis3L2 homolog is required for cytokinesis from polyploid “titan” growth stages. This phenotype of C. neoformans Ssd1/Dis3L2 deletion is consistent with those of inactive fungal pseudonucleases, yet the protein retains an active site sequence signature. We propose that a nuclease-independent function for Dis3L2 arose in an ancestral hyphae-forming fungus. This second function has been conserved across hundreds of millions of years, whereas the RNase activity was lost repeatedly in independent lineages.     

Primary and secondary siRNA synthesis triggered by RNAs from food bacteria in the ciliate Paramecium tetraurelia

In various organisms, an efficient RNAi response can be triggered by feeding cells with bacteria producing double-stranded RNA (dsRNA) against an endogenous gene. However, the detailed mechanisms and natural functions of this pathway are not well understood in most cases. Here, we studied siRNA biogenesis from exogenous RNA and its genetic overlap with endogenous RNAi in the ciliate Paramecium tetraurelia by high-throughput sequencing. Using wild-type and mutant strains deficient for dsRNA feeding we found that high levels of primary siRNAs of both strands are processed from the ingested dsRNA trigger by the Dicer Dcr1, the RNA-dependent RNA polymerases Rdr1 and Rdr2 and other factors. We further show that this induces the synthesis of secondary siRNAs spreading along the entire endogenous mRNA, demonstrating the occurrence of both 3′-to-5′ and 5′-to-3′ transitivity for the first time in the SAR clade of eukaryotes (Stramenopiles, Alveolates, Rhizaria). Secondary siRNAs depend on Rdr2 and show a strong antisense bias; they are produced at much lower levels than primary siRNAs and hardly contribute to RNAi efficiency. We further provide evidence that the Paramecium RNAi machinery also processes single-stranded RNAs from its bacterial food, broadening the possible natural functions of exogenously induced RNAi in this organism.     

The Making of an siRNA

An array of gene silencing pathways share a common early step: Dicer cleaves double-stranded RNA (dsRNA) into approximately 20-25 nucleotide fragments that direct the silencing machinery to specific targets. A recent report in Cell reveals how Dicer's two RNase III domains collaborate during dsRNA processing and sets the stage for a deeper understanding of Dicer's roles in later phases of silencing complex assembly.