Schistosoma mansoni: the dicer gene and its expression

RNA interference (RNAi) is a gene silencing mechanism that plays an important role in regulating gene expression in many eukaryotes and has become a valuable molecular tool for analyzing gene function. Multi-domain nucleases called Dicer proteins play pivotal roles in RNAi. In this paper, we characterize the structure and expression of the Dicer gene from the platyhelminth parasite Schistosoma mansoni. The gene (SmDicer) is over 54kb long and comprises 30 exons that potentially encode a 2641 amino acid protein. This is the largest Dicer protein yet described. SmDicer contains all domains that are characteristic of metazoan dicers including an amino terminal helicase domain, DUF283, a PAZ domain, two RNAse III domains and an RNA binding domain. An examination of the available S. mansoni genome sequence suggests that the Dicer gene described here is the only Dicer gene in the parasite genome. SmDicer is expressed throughout schistosome development suggesting that RNAi technologies might be employed in deciphering gene function in all life stages of this parasite.     

Structure of the Arabidopsis thaliana DCL4 DUF283 domain reveals a noncanonical double-stranded RNA-binding fold for protein–protein interaction

Dicer or Dicer-like (DCL) protein is a catalytic component involved in microRNA (miRNA) or small interference RNA (siRNA) processing pathway, whose fragment structures have been partially solved. However, the structure and function of the unique DUF283 domain within dicer is largely unknown. Here we report the first structure of the DUF283 domain from the Arabidopsis thaliana DCL4. The DUF283 domain adopts an α-β-β-β-α topology and resembles the structural similarity to the double-stranded RNA-binding domain. Notably, the N-terminal α helix of DUF283 runs cross over the C-terminal α helix orthogonally, therefore, N- and C-termini of DUF283 are in close proximity. Biochemical analysis shows that the DUF283 domain of DCL4 displays weak dsRNA binding affinity and specifically binds to double-stranded RNA-binding domain 1 (dsRBD1) of Arabidopsis DRB4, whereas the DUF283 domain of DCL1 specifically binds to dsRBD2 of Arabidopsis HYL1. These data suggest a potential functional role of the Arabidopsis DUF283 domain in target selection in small RNA processing.     

Dicer 的结构功能及其与肿瘤的关系

Dicer蛋白是RNaseⅢ家族中重要的一员,对miRNA或siRNA的产生起着至关重要的作用。Dicer蛋白通常由1个DEXH盒子或H盒子、1个DUF283结构域、1个PAZ结构域、2个RNaseⅢ结构域(RNaseⅢa和RNaseⅢb)和1个dsRNA结合结构域组成。Dicer蛋白的分子结构决定了其在miRNAs合成中发挥着重要作用。Dicer及生成的miRNA与肿瘤又有着密切关系。本文主要针对Dicer及其与肿瘤的关系作简要综述。     

SCOOP: a simple method for identification of novel protein superfamily relationships

MOTIVATION: Profile searches of sequence databases are a sensitive way to detect sequence relationships. Sophisticated profile-profile comparison algorithms that have been recently introduced increase search sensitivity even further. RESULTS: In this article, a simpler approach than profile-profile comparison is presented that has a comparable performance to state-of-the-art tools such as COMPASS, HHsearch and PRC. This approach is called SCOOP (Simple Comparison Of Outputs Program), and is shown to find known relationships between families in the Pfam database as well as detect novel distant relationships between families. Several novel discoveries are presented including the discovery that a domain of unknown function (DUF283) found in Dicer proteins is related to double-stranded RNA-binding domains. AVAILABILITY: SCOOP is freely available under a GNU GPL license from http://www.sanger.ac.uk/Users/agb/SCOOP/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

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.     

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.     

Structural and Functional Characterization of DUF1471 Domains of Salmonella Proteins SrfN,YdgH/SssB,and YahO

Bacterial species in the Enterobacteriaceae typically contain multiple paralogues of a small domain of unknown function (DUF1471) from a family of conserved proteins also known as YhcN or BhsA/McbA. Proteins containing DUF1471 may have a single or three copies of this domain. Representatives of this family have been demonstrated to play roles in several cellular processes including stress response, biofilm formation, and pathogenesis. We have conducted NMR and X-ray crystallographic studies of four DUF1471 domains from Salmonella representing three different paralogous DUF1471 subfamilies: SrfN, YahO, and SssB/YdgH (two of its three DUF1471 domains: the N-terminal domain I (residues 21–91), and the C-terminal domain III (residues 244–314)). Notably, SrfN has been shown to have a role in intracellular infection by Salmonella Typhimurium. These domains share less than 35% pairwise sequence identity. Structures of all four domains show a mixed α+β fold that is most similar to that of bacterial lipoprotein RcsF. However, all four DUF1471 sequences lack the redox sensitive cysteine residues essential for RcsF activity in a phospho-relay pathway, suggesting that DUF1471 domains perform a different function(s). SrfN forms a dimer in contrast to YahO and SssB domains I and III, which are monomers in solution. A putative binding site for oxyanions such as phosphate and sulfate was identified in SrfN, and an interaction between the SrfN dimer and sulfated polysaccharides was demonstrated, suggesting a direct role for this DUF1471 domain at the host-pathogen interface.     

The Role of Human Dicer-dsRBD in Processing Small Regulatory RNAs

One of the most exciting recent developments in RNA biology has been the discovery of small non-coding RNAs that affect gene expression through the RNA interference (RNAi) mechanism. Two major classes of RNAs involved in RNAi are small interfering RNA (siRNA) and microRNA (miRNA). Dicer, an RNase III enzyme, plays a central role in the RNAi pathway by cleaving precursors of both of these classes of RNAs to form mature siRNAs and miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). miRNA and siRNA precursors are quite structurally distinct; miRNA precursors are short, imperfect hairpins while siRNA precursors are long, perfect duplexes. Nonetheless, Dicer is able to process both. Dicer, like the majority of RNase III enzymes, contains a dsRNA binding domain (dsRBD), but the data are sparse on the exact role this domain plays in the mechanism of Dicer binding and cleavage. To further explore the role of human Dicer-dsRBD in the RNAi pathway, we determined its binding affinity to various RNAs modeling both miRNA and siRNA precursors. Our study shows that Dicer-dsRBD is an avid binder of dsRNA, but its binding is only minimally influenced by a single-stranded – double-stranded junction caused by large terminal loops observed in miRNA precursors. Thus, the Dicer-dsRBD contributes directly to substrate binding but not to the mechanism of differentiating between pre-miRNA and pre-siRNA. In addition, NMR spin relaxation and MD simulations provide an overview of the role that dynamics contribute to the binding mechanism. We compare this current study with our previous studies of the dsRBDs from Drosha and DGCR8 to give a dynamic profile of dsRBDs in their apo-state and a mechanistic view of dsRNA binding by dsRBDs in general.     

Synthetic shRNAs as potent RNAi triggers

Designing potent silencing triggers is key to the successful application of RNA interference (RNAi) in mammals. Recent studies suggest that the assembly of RNAi effector complexes is coupled to Dicer cleavage. Here we examine whether transfection of optimized Dicer substrates results in an improved RNAi response. Dicer cleavage of chemically synthesized short hairpin RNAs (shRNAs) with 29-base-pair stems and 2-nucleotide 3' overhangs produced predictable homogeneous small RNAs comprising the 22 bases at the 3' end of the stem. Consequently, direct comparisons of synthetic small interfering RNAs and shRNAs that yield the same small RNA became possible. We found synthetic 29-mer shRNAs to be more potent inducers of RNAi than small interfering RNAs. Maximal inhibition of target genes was achieved at lower concentrations and silencing at 24 h was often greater. These studies provide the basis for an improved approach to triggering experimental silencing via the RNAi pathway.     

Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.     

The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes

RISC, the RNA-induced silencing complex, uses short interfering RNAs (siRNAs) or micro RNAs (miRNAs) to select its targets in a sequence-dependent manner. Key RISC components are Argonaute proteins, which contain two characteristic domains, PAZ and PIWI. PAZ is highly conserved and is found only in Argonaute proteins and Dicer. We have solved the crystal structure of the PAZ domain of Drosophila Argonaute2. The PAZ domain contains a variant of the OB fold, a module that often binds single-stranded nucleic acids. PAZ domains show low-affinity nucleic acid binding, probably interacting with the 3' ends of single-stranded regions of RNA. PAZ can bind the characteristic two-base 3' overhangs of siRNAs, indicating that although PAZ may not be a primary nucleic acid binding site in Dicer or RISC, it may contribute to the specific and productive incorporation of siRNAs and miRNAs into the RNAi pathway.     

RNAi induction and activation in mammalian muscle cells where Dicer and eIF2C translation initiation factors are barely expressed

Dicer plays an important role in the course of RNA interference (RNAi), i.e., it digests long double-stranded RNAs into 21-25 nucleotide small-interfering RNA (siRNA) duplexes functioning as sequence-specific RNAi mediators. In this study, we investigated the expression levels of Dicer and eIF2C1 approximately 4, which, like Dicer, appear to participate in mammalian RNAi, in various mouse tissues. Results indicate that the levels of eIF2C1 approximately 4 as well as Dicer are lower in skeletal muscle and heart than in other tissues. To see if RNAi could occur under such a condition with low levels of expression of Dicer and eIF2C1 approximately 4, we examined RNAi activity in mouse skeletal muscle fibers. The results indicate that RNAi can be induced by synthetic siRNA duplexes in muscle fibers. We further examined RNAi activity during myogenic differentiation of mouse C2C12 cells. The data indicate that although the expression levels of Dicer and eIF2C1 approximately 4 decrease during the differentiation, RNAi can be induced in the cells. Altogether, the data presented here suggest that muscle cells retain the ability to induce RNAi, although Dicer and eIF2C1 approximately 4 appear to be barely expressed in them.     

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。     

RNA诱导沉默复合体中的生物大分子及其装配

在RNA干扰机制中,双链RNA诱导同源RNA降解的过程依赖于RNA诱导沉默复合体（RISC）的活性。RISC由Dicer酶,Argonaute蛋白,siRNA等多种生物大分子装配而成,对这些大分子的结构和功能进行深入细致的研究,有助于进一步了解RISC的形成过程、作用方式,以及阐明整个RNAi过程的作用机制。研究表明,RISC中的Dicer具有RNaseIII结构域,在RNAi的起始阶段负责催化siRNA的产生,在RISC装配过程中起稳定RISC中间体结构和功能的作用;Argonaute蛋白是RISC中的核心蛋白,有PAZ和PIWI两个主要的结构域,前者为siRNA的传递提供结合位点,后者是RISC中的酶切割活性中心;siRNA是RISC完成特异性切割作用的向导,在成熟的RISC中虽然只包含siRNA的一条链,但siRNA在RISC形成过程中的双链结构是保证RNAi效应的决定因素。尽管RISC中还存在其他一些功能未知的蛋白质,但在RISC组分结构及功能研究方面取得的进展为建立一个可能的RISC装配模型提供了理论基础。     

Backbone resonance assignments of the monomeric DUF59 domain of human Fam96a

Proteins containing a domain of unknown function 59 (DUF59) appear to have a variety of physiological functions, ranging from iron-sulfur cluster assembly to DNA repair. DUF59 proteins have been found in bacteria, archaea and eukaryotes, however Fam96a and Fam96b are the only mammalian proteins predicted to contain a DUF59 domain. Fam96a is an 18 kDa protein comprised primarily of a DUF59 domain (residues 31–157) and an N-terminal signal peptide (residues 1–27). Interestingly, the DUF59 domain of Fam96a exists as monomeric and dimeric forms in solution, and X-ray crystallography studies of both forms unexpectedly revealed two different domain-swapped dimer structures. Here we report the backbone resonance assignments and secondary structure of the monomeric form of the 127 residue DUF59 domain of human Fam96a. This study provides the basis for further understanding the structural variability exhibited by Fam96a and the mechanism for domain swapping.     

Dicer has a crucial role in the early stage of adipocyte differentiation, but not in lipid synthesis, in 3T3-L1 cells

Dicer is a rate-limiting enzyme for microRNA (miRNA) synthesis. To determine the effects of Dicer on adipogenesis, we performed stage-specific knockdown of Dicer using adenovirus encoding short-hairpin RNAi against Dicer in 3T3-L1 cells. When cells were infected with the adenovirus before induction of adipocyte differentiation, Dicer RNAi suppressed the gene expression of inducers of adipocyte differentiation such as PPARγ, C/EBPα, and FAS in 3T3-L1 cells during adipocyte differentiation. Concurrently, both adipocyte differentiation and cellular lipid accumulation were cancelled by Dicer RNAi when compared with control RNAi. Meanwhile, we addressed the roles of Dicer in lipid synthesis and accumulation in the final stages of differentiation. When the differentiated cells at day 4 after induction of differentiation were infected with adenovirus Dicer RNAi, cellular lipid accumulation was unchanged. Consistent with this, Dicer RNAi had no effects on the expression of genes related to cellular lipid accumulation, including PPARγ and FAS. Thus, Dicer controls proadipogenic genes such as C/EBPα and PPARγ in the early, but not in the late, stage of adipogenesis via regulation of miRNA synthesis.     

RNase III enzymes and the initiation of gene silencing

Our understanding of RNA interference has been enhanced by new data concerning RNase III molecules. The role of Dicer has previously been established in RNAi as the originator of 22-mers characteristic of silencing phenomena. Recently, a related RNAse III enzyme, Drosha, has surfaced as another component of the RNAi pathway. In addition to biochemistry, protein structures have proven to be helpful in deciphering the enzymology of RNase III molecules.