Ribonuclease Dicer Research Articles

MicroRNAs and cancer: past, present, and potential future

MicroRNAs (miRNAs) are a class of small RNAs that have revealed a new level of gene regulation in the cell. After being processed by Drosha and Dicer RNase III endonucleases, mature miRNAs can inhibit the translation of mRNA by directing a RNA-induced silencing complex (RISC) to the target mRNA. miRNAs are making an impact in our understanding of cancer biology. Acting as either tumor suppressors or oncogenes, miRNAs regulate several genes known to play important roles in cancer. With the discovery of miRNAs comes the need for new techniques to study their activity. Bioinformatic tools can be used to predict mRNA targets of miRNA, but validation of miRNA regulation of predicted targets is imperative. miRNAs are differentially expressed in normal and tumor cells as well as between tumor subtypes. These differences may be useful as prognostic and predictive markers in cancer patients. The study of miRNAs holds much promise for improving diagnosis and treatment of cancer.

Virus meets RNAi

The European Science Foundation (ESF)–European Molecular Biology Organization (EMBO) Symposium on Antiviral Applications of RNA Interference took place between 5 and 10 April 2008, in Sant Feliu de Guixols, Spain, and was organized by J. Kurreck and B. Berkhout. ![][1] Small RNAs are important regulators of gene expression that operate through a range of RNA‐silencing mechanisms. Of these, RNA interference (RNAi)—gene silencing through small‐interfering RNAs (siRNAs) derived from exogenous double‐stranded RNA (dsRNA)—and the microRNA (miRNA) pathway—gene silencing guided by small RNAs encoded in the genome—are the best known. The discovery that dsRNA induces RNAi (Fire et al , 1998) and the appreciation of its antiviral activity revolutionized our view of viruses in many ways. The European Science Foundation (ESF)–European Molecular Biology Organization (EMBO) Symposium on Antiviral Applications of RNA Interference brought together about 50 scientists working at the interface between virology and RNAi. Here, I review the main issues that were discussed at the meeting. RNAi provides an antiviral defence mechanism in plants, nematodes and insects. The cleavage of viral dsRNA by the ribonuclease Dicer generates viral siRNAs (v‐siRNAs), which are incorporated into the RNA‐induced silencing complex (RISC) and direct RISC activity onto messenger RNAs (mRNAs) in a sequence‐specific manner. Recognition of a complementary sequence then triggers the endonucleic cleavage (slicer activity) or translational inhibition of the viral target RNAs by an Argonaute family member (Fig 1). Figure 1. RNA silencing in virus infections. ( A ) RNA interference (RNAi) as an antiviral defence mechanism. Viruses might produce double‐stranded RNAs (dsRNAs) that are processed by the RNAi machinery and thereby programme the RNA‐induced silencing complex (RISC) to degrade viral RNAs. ( B ) Cellular or viral microRNAs (miRNAs), or viral RNAs with structural similarity to pre‐miRNAs, regulate viral and host gene expression, and contribute to viral pathogenesis. miRISC, microRNA‐induced silencing complex; pre‐miRNA, precursor miRNA; pri‐miRNA, primary miRNA … [1]: /embed/graphic-1.gif

A feedback loop comprising lin-28 and let-7 controls pre-let-7 maturation during neural stem-cell commitment

miRNA populations, including mammalian homologues of lin-4 (mir-125) and let-7, undergo a marked transition during stem-cell differentiation. Originally identified on the basis of their mutational phenotypes in stem-cell maturation, mir-125 and let-7 are strongly induced during neural differentiation of embryonic stem (ES) cells and embryocarcinoma (EC) cells. We report that embryonic neural stem (NS) cells express let-7 and mir-125, and investigate post-transcriptional mechanisms contributing to the induction of let-7. We demonstrate that the pluripotency factor Lin-28 binds the pre-let-7 RNA and inhibits processing by the Dicer ribonuclease in ES and EC cells. In NS cells, Lin-28 is downregulated by mir-125 and let-7, allowing processing of pre-let-7 to proceed. Suppression of let-7 or mir-125 activity in NS cells led to upregulation of Lin-28 and loss of pre-let-7 processing activity, suggesting that let-7, mir-125 and lin-28 participate in an autoregulatory circuit that controls miRNA processing during NS-cell commitment.

Biogenesis of Short Intronic Repeat 27-Nucleotide Small RNA from Endothelial Nitric-oxide Synthase Gene

Endothelial nitric-oxide synthase (eNOS) is a constitutively expressed gene in endothelium that produces NO and is critical for vascular integrity. Previously, we reported that the 27-nucleotide (nt) repeat polymorphism in eNOS intron 4, a source of 27-nt small RNA, which inhibits eNOS expression, were associated with cardiovascular risk and expression of the eNOS gene. In the current study, we investigated the biogenesis of the intron 4-derived 27-nt small RNA. Using Northern blot, we showed that the eNOS-derived 27-nt short intronic repeat RNA (sir-RNA) expressed only in the eNOS expressing endothelial cells. Cells containing 10 x 27- or 5 x 27-nt repeats produced higher levels of 27nt sir-RNA and lower levels of eNOS mRNA than the cells with 4 x 27-nt repeats. The 27nt sir-RNA was mostly present within the endothelial nuclei. When the splicing junctions of the 27-nt repeat containing intron 4 in the full-length eNOS cDNA vector were mutated, 27nt sir-RNA biogenesis was abolished. Suppression of Drosha or Dicer diminished the biogenesis of the 27nt sir-RNA. Our study suggests that the 27nt sir-RNA derived through eNOS pre-mRNA splicing may represent a new class of small RNA. The more eNOS is transcribed or higher number of the 27-nt repeats, the more 27nt sir-RNA is produced, which functions as a negative feedback self-regulator by specifically inhibiting the host gene eNOS expression. This novel molecular model may be responsible for quantitative differences between individuals carrying different numbers of the polymorphic repeats hence the cardiovascular risk.

RNA interference

RNA interference

Structural and biochemical insights into the dicing mechanism of mouse Dicer: A conserved lysine is critical for dsRNA cleavage

Dicer, an RNase III enzyme, initiates RNA interference by processing precursor dsRNAs into mature microRNAs and small-interfering RNAs. It is also involved in loading and activation of the RNA-induced silencing complex. Here, we report the crystal structures of a catalytically active fragment of mouse Dicer, containing the RNase IIIb and dsRNA binding domains, in its apo and Cd(2+)-bound forms, at 1.68- and 2.8-A resolution, respectively. Models of this structure with dsRNA reveal that a lysine residue, highly conserved in Dicer RNase IIIa and IIIb domains and in Drosha RNase IIIb domains, has the potential to participate in the phosphodiester bond cleavage reaction by stabilizing the transition state and leaving group of the scissile bond. Mutational and enzymatic assays confirm the importance of this lysine in dsRNA cleavage, suggesting that this lysine represents a conserved catalytic residue of Dicers. The structures also reveals a approximately 45-aa region within the RNase IIIb domain that harbors an alpha-helix at the N-terminal half and a flexible loop at the C-terminal half, features not present in previously reported structures of homologous RNase III domains from either bacterial RNase III enzymes or Giardia Dicer. N-terminal residues of this alpha-helix have the potential to engage in minor groove interaction with dsRNA substrates.

Heterochromatin and RNAi Are Required to Establish CENP-A Chromatin at Centromeres

Heterochromatin is defined by distinct posttranslational modifications on histones, such as methylation of histone H3 at lysine 9 (H3K9), which allows heterochromatin protein 1 (HP1)-related chromodomain proteins to bind. Heterochromatin is frequently found near CENP-A chromatin, which is the key determinant of kinetochore assembly. We have discovered that the RNA interference (RNAi)-directed heterochromatin flanking the central kinetochore domain at fission yeast centromeres is required to promote CENP-A(Cnp1) and kinetochore assembly over the central domain. The H3K9 methyltransferase Clr4 (Suv39); the ribonuclease Dicer, which cleaves heterochromatic double-stranded RNA to small interfering RNA (siRNA); Chp1, a component of the RNAi effector complex (RNA-induced initiation of transcriptional gene silencing; RITS); and Swi6 (HP1) are required to establish CENP-A(Cnp1) chromatin on naïve templates. Once assembled, CENP-A(Cnp1) chromatin is propagated by epigenetic means in the absence of heterochromatin. Thus, another, potentially conserved, role for centromeric RNAi-directed heterochromatin has been identified.

Coupling of Double-Stranded RNA Synthesis and siRNA Generation in Fission Yeast RNAi

The fission yeast centromeric repeats are transcribed and ultimately processed into small interfering RNAs (siRNAs) that are required for heterochromatin formation. siRNA generation requires dsRNA synthesis by the RNA-directed RNA polymerase complex (RDRC) and processing by the Dicer ribonuclease. Here we show that Dcr1, the fission yeast Dicer, is physically associated with RDRC. Dcr1 generates siRNAs in an ATP-dependent manner that requires its conserved N-terminal helicase domain. Furthermore, C-terminal truncations of Dcr1 that abolish its interaction with RDRC, but can generate siRNA in vitro, abolish siRNA generation and heterochromatic gene silencing in vivo. Finally, reconstitution experiments show that the association of Dcr1 with RDRC strongly stimulates the dsRNA synthesis activity of RDRC. Our results suggest that heterochromatic dsRNA synthesis and siRNA generation are physically coupled processes. This coupling has implications for cis-restriction of siRNA-mediated heterochromatin assembly and for mechanisms that give rise to siRNA strand polarity.

Suppression of MicroRNA-Silencing Pathway by HIV-1 During Virus Replication

MicroRNAs (miRNAs) are single-stranded noncoding RNAs of 19 to 25 nucleotides that function as gene regulators and as a host cell defense against both RNA and DNA viruses. We provide evidence for a physiological role of the miRNA-silencing machinery in controlling HIV-1 replication. Type III RNAses Dicer and Drosha, responsible for miRNA processing, inhibited virus replication both in peripheral blood mononuclear cells from HIV-1-infected donors and in latently infected cells. In turn, HIV-1 actively suppressed the expression of the polycistronic miRNA cluster miR-17/92. This suppression was found to be required for efficient viral replication and was dependent on the histone acetyltransferase Tat cofactor PCAF. Our results highlight the involvement of the miRNA-silencing pathway in HIV-1 replication and latency.

Ribonuclease Dicer Cleaves Triplet Repeat Hairpins into Shorter Repeats that Silence Specific Targets

Ribonuclease Dicer functions in cells to excise microRNAs from their precursors and process long double-stranded RNAs into short interfering RNAs. We show that transcripts containing long hairpin structures composed of CNG repeats are another class of Dicer targets. The cellular levels of transcripts from mutant genes involved in triplet repeat expansion diseases such as myotonic dystrophy type 1, Huntington's disease, and spinocerebellar ataxia type 1 are under Dicer control. The Dicer-induced downregulation of the mutant transcript in myotonic dystrophy cells is accompanied by the downregulation of transcripts containing long complementary repeats. Short CUG repeats generated from long repeat hairpins act as siRNAs and use the RNA interference pathway to trigger the downstream silencing effects. We demonstrate that synthetic oligonucleotides composed of repeats are highly specific in the silencing of mutant transcripts containing complementary repeats and may be considered as potential therapeutic agents.

S06A5 Dicer RNase IIIドメインの結晶構造とdsRNA切断活性(ncRNAのプロセスと機能に関わる構造生物学,シンポジウム,第45回日本生物物理学会年会)

S06A5 Dicer RNase IIIドメインの結晶構造とdsRNA切断活性(ncRNAのプロセスと機能に関わる構造生物学,シンポジウム,第45回日本生物物理学会年会)

Structural Basis for Double-Stranded RNA Processing by Dicer

The specialized ribonuclease Dicer initiates RNA interference by cleaving double-stranded RNA (dsRNA) substrates into small fragments about 25 nucleotides in length. In the crystal structure of an intact Dicer enzyme, the PAZ domain, a module that binds the end of dsRNA, is separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface. The 65 angstrom distance between the PAZ and RNase III domains matches the length spanned by 25 base pairs of RNA. Thus, Dicer itself is a molecular ruler that recognizes dsRNA and cleaves a specified distance from the helical end.

MiRNA and Dicer in the mammalian lens: expression of brain-specific miRNAs in the lens

Micro RNAs (miRNAs) are approximately 22 nucleotide molecules that regulate gene expression post-transcriptionally and govern a wide range of physiological and developmental processes. Evidence now indicates that miRNAs can also coordinately down-regulate transcript levels for very large groups of genes in a tissue-specific manner, in addition to their ability to suppress protein translation. Here, we examine expression of specific miRNAs and Dicer ribonuclease that is required for miRNA biogenesis in mouse and rat lenses. Northern blot analysis demonstrated lens expression of brain-specific miR-124 and miR-7 in lenses, as well as miR-125b and let-7a. In addition, we provide evidence that muscle specific miR-1 is not present in lens. We detected Dicer transcripts in 21 day, 6 week, and 1 year mouse lenses and 15 day rat lens, and detected Dicer protein in adult lens protein samples. Immunohistochemical examination of late embryonic, post-natal, and adult rat lens sections identified expression of Dicer in differentiating fiber cells that undergo pronounced cell elongation in the lens interior and anterior epithelial cells. The present study provides evidence that miRNAs, which include brain-specific forms, and Dicer are expressed in mammalian lenses, indicating that fundamental aspects of miRNA biology are utilized by the lens during late embryonic and post-natal development and in adult lenses.

Dicer‐Derived MicroRNAs Are Utilized by the Fragile X Mental Retardation Protein for Assembly on Target RNAs

In mammalian cells, fragile X mental retardation protein (FMRP) has been reported to be part of a microRNA (miRNA)-containing effector ribonucleoprotien (RNP) complex believed to mediate translational control of specific mRNAs. Here, using recombinant proteins, we demonstrate that human FMRP can act as a miRNA acceptor protein for the ribonuclease Dicer and facilitate the assembly of miRNAs on specific target RNA sequences. The miRNA assembler property of FMRP was abrogated upon deletion of its single-stranded (ss) RNA binding K-homology domains. The requirement of FMRP for efficient RNA interference (RNAi) in vivo was unveiled by reporter gene silencing assays using various small RNA inducers, which also supports its involvement in an ss small interfering RNA (siRNA)-containing RNP (siRNP) effector complex in mammalian cells. Our results define a possible role for FMRP in RNA silencing and may provide further insight into the molecular defects in patients with the fragile X syndrome.

MicroRNAs Regulate Brain Morphogenesis in Zebrafish

MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, we generated maternal-zygotic dicer (MZdicer) mutants that disrupt the Dicer ribonuclease III and double-stranded RNA-binding domains. Mutant embryos do not process precursor miRNAs into mature miRNAs, but injection of preprocessed miRNAs restores gene silencing, indicating that the disrupted domains are dispensable for later steps in silencing. MZdicer mutants undergo axis formation and differentiate multiple cell types but display abnormal morphogenesis during gastrulation, brain formation, somitogenesis, and heart development. Injection of miR-430 miRNAs rescues the brain defects in MZdicer mutants, revealing essential roles for miRNAs during morphogenesis.

Small RNA-mediated chromatin modification and transcriptional gene silencing

Small RNAs, such as short-interfering RNAs (siRNAs) and microRNAs (miRNAs), regulate gene expression in a sequence-dependent manner in animals and plants. These small RNAs are generated from long double-stranded (ds) RNAs or pre-miRNAs by the ribonuclease Dicer and become incorporated into RNA interference (RNAi)-induced silencing complexes (RISCs) or miRNA ribonucleoprotein complexes (miRNPs). These complexes have the ability to cleave target mRNAs with perfect base-pairing and inhibit the translation of target mRNAs with partial base-pairing. In addition, small RNAs can also act on the chromosomes in the nucleus. In this process, it is known that siRNAs targeted to CpG islands within promoters can induce RNA-directed DNA methylation and play a role in heterochromatic gene silencing. Thus, small RNAs can regulate gene expression at both the transcriptional and post-transcriptional levels. In this review, we focus on small RNA-mediated transcriptional gene silencing and on its role and potential therapeutic applications in chromosome maintenance and gene regulation, including epigenetic regulation.

Two RNAi Complexes, RITS and RDRC, Physically Interact and Localize to Noncoding Centromeric RNAs

RNAi-mediated heterochromatin assembly in fission yeast requires the RNA-induced transcriptional silencing (RITS) complex and a putative RNA-directed RNA polymerase (Rdp1). Here we show that Rdp1 is associated with two conserved proteins, Hrr1, an RNA helicase, and Cid12, a member of the polyA polymerase family, in a complex that has RNA-directed RNA polymerase activity (RDRC, RNA-directed RNA polymerase complex). RDRC physically interacts with RITS in a manner that requires the Dicer ribonuclease (Dcr1) and the Clr4 histone methyltransferase. Moreover, both complexes are localized to the nucleus and associate with noncoding centromeric RNAs in a Dcr1-dependent manner. In cells lacking Rdp1, Hrr1, or Cid12, RITS complexes are devoid of siRNAs and fail to localize to centromeric DNA repeats to initiate heterochromatin assembly. These findings reveal a physical and functional link between Rdp1 and RITS and suggest that noncoding RNAs provide a platform for siRNA-dependent localization of RNAi complexes to specific chromosome regions.

Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microrna in host gene expression.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by the ribonuclease Dicer from distinct precursors, double-stranded RNA (dsRNA) and hairpin RNAs, respectively, although either may guide RNA silencing via a similar complex. The siRNA pathway is antiviral, whereas an emerging role for miRNAs is in the control of development. Here, we describe a virulence factor encoded by turnip yellow mosaic virus, p69, which suppresses the siRNA pathway but promotes the miRNA pathway in Arabidopsis thaliana. p69 suppression of the siRNA pathway is upstream of dsRNA and is as effective as genetic mutations in A. thaliana genes involved in dsRNA production. Possibly as a consequence of p69 suppression, p69-expressing plants contained elevated levels of a Dicer mRNA and of miRNAs as well as a correspondingly enhanced miRNA-guided cleavage of two host mRNAs. Because p69-expressing plants exhibited disease-like symptoms in the absence of viral infection, our findings suggest a novel mechanism for viral virulence by promoting the miRNA-guided inhibition of host gene expression.

Characterization of the interactions between mammalian PAZ PIWI domain proteins and Dicer.

PAZ PIWI domain (PPD) proteins, together with the RNA cleavage products of Dicer, form ribonucleoprotein complexes called RNA-induced silencing complexes (RISCs). RISCs mediate gene silencing through targeted messenger RNA cleavage and translational suppression. The PAZ domains of PPD and Dicer proteins were originally thought to mediate binding between PPD proteins and Dicer, although no evidence exists to support this theory. Here we show that PAZ domains are not required for PPD protein-Dicer interactions. Rather, a subregion of the PIWI domain in PPD proteins, the PIWI-box, binds directly to the Dicer RNase III domain. Stable binding between PPD proteins and Dicer was dependent on the activity of Hsp90. Unexpectedly, binding of PPD proteins to Dicer inhibits the RNase activity of this enzyme in vitro. Lastly, we show that PPD proteins and Dicer are present in soluble and membrane-associated fractions, indicating that interactions between these two types of proteins may occur in multiple compartments.

RNAi-mediated targeting of heterochromatin by the RITS complex.

RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci.