Dicer-1: Biological Overview | Evolutionary Homologs | Regulation & Characterization of RNAi Process | Developmental Biology | Effects of RNAi Depletion | References
Gene name - Dicer-1

Synonyms -

Cytological map position - 94C4

Function - enzyme

Keywords - double-stranded RNA interference (RNAi), post-transcriptional gene regulation (PTGS)

Symbol - Dcr-1

FlyBase ID: FBgn0039016

Genetic map position -

Classification - ribonuclease III family, double-stranded RNA binding domain, DEAD/DEAH box helicase, Fibronectin type III domain

Cellular location - presumably cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene | UniGene |
Recent literature
Jakob, L., Treiber, T., Treiber, N., Gust, A., Kramm, K., Hansen, K., Stotz, M., Wankerl, L., Herzog, F., Hannus, S., Grohmann, D. and Meister, G. (2016). Structural and functional insights into the fly microRNA biogenesis factor Loquacious. RNA [Epub ahead of print]. PubMed ID: 26769856
Summary:
In the microRNA (miRNA) pathway, Dicer processes precursors to mature miRNAs. For efficient processing, double-stranded RNA-binding proteins support Dicer proteins. In flies, Loquacious (Loqs) interacts with Dicer1 (Dcr1) to facilitate miRNA processing. The structure of the third double-stranded RNA-binding domain (dsRBD) of Loqs has been solved, and specific structural elements have been defined that interact with Dcr1. In addition, the linker preceding dsRBD3 was shown to contribute significantly to Dcr1 binding. Furthermore, this structural work demonstrates that the third dsRBD of Loqs forms homodimers. Mutations in the dimerization interface abrogate Dcr1 interaction. Loqs, however, binds to Dcr1 as a monomer using the identified dimerization surface, suggesting that Loqs might form dimers under conditions where Dcr1 is absent or not accessible. Since critical sequence elements are conserved, it is suggested that dimerization might be a general feature of dsRBD proteins in gene silencing.
Banerjee, A. and Roy, J.K. (2017). Dicer-1 regulates proliferative potential of Drosophila larval neural stem cells through bantam miRNA based down-regulation of the G1/S inhibitor Dacapo. Dev Biol [Epub ahead of print]. PubMed ID: 28109717
Summary:
This study elucidates the role of miRNA in cell cycle regulation during brain development in Drosophila. It was found that lineage specific depletion of dicer-1, a classically acknowledged miRNA biogenesis protein in neuroblasts leads to a reduction in their numbers and size in the third instar larval central brain. These brains also show lower number of mitotically active cells and when homozygous mitotic clones were generated in an otherwise heterozygous dicer-1 mutant background via MARCM technique, they show reduced number of progeny cells in individual clones, substantiating the adverse effect of the loss of dicer-1 on the proliferative potential of neuroblasts. bantam miRNA, which has been classically reported to be involved in tissue growth was found to be expressed in neuroblasts and undergo reduced expression in Dicer-1 depleted background in the third instar larval brain. Reduction in the number and proliferative potential of neuroblasts in bantam mutant background implies a pivotal role played by bantam miRNA in maintenance of neuroblast number. Since in both Dicer-1 and bantam depleted genetic backgrounds, Dacapo, an inhibitor of cyclin E-Cdk complex, was found to have elevated expression, the study postulates a molecular mechanism involving bantam-Dacapo-Cyclin E/Cdk complex that regulates the G1-S phase transition of Drosophila neuroblasts.


BIOLOGICAL OVERVIEW

Because of its ability to digest double-stranded RNA (dsRNA) into uniformly sized, small RNAs, this enzyme has been named Dicer. Dicer contains a region of homology to the RDE1/QDE2/ARGONAUTE family that has been genetically linked to RNAi (RNA interference). RNAi is a mechanism through which double-stranded RNAs silence cognate genes. In plants and animals, this can occur at both the transcriptional and the post-transcriptional levels. In both plants and animals, RNAi is characterized by the presence of RNAs of about 22 nucleotides in length that are homologous to the gene that is being suppressed. These 22-nucleotide sequences serve as guide sequences that instruct a multicomponent nuclease, RISC (RNA induced silencing complex), to destroy specific messenger RNAs. Dicer, an enzyme that can produce putative guide RNAs, is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs, and is evolutionarily conserved in worms, flies, plants, fungi and mammals. The enzyme has a distinctive structure, which includes a helicase domain (involved in unwinding RNA) and dual RNase III motifs (Bernstein, 2001).

Post-transcriptional gene silencing (PTGS) can be distinguished from RNAi by the fact that the target mRNA is not degraded but is translationally silenced. Biochemical studies have suggested that PTGS is accomplished, in part, by a mechanism similar to RNAi in that involves a nuclease that targets RNAs for degradation. Subsequently, an enzyme complex similar to RISC, targets mRNA for translational silencing. The specificity of this complex may derive from the incorporation of a small guide sequence that is homologous to the mRNA substrate. These ~22-nucleotide RNAs, originally identified in plants that were actively silencing transgenes, have been produced during RNAi in vitro using an extract prepared from Drosophila embryos. Putative guide RNAs can also be produced in extracts from Drosophila S2 cells. To investigate the mechanism of RNAi and PTGS, both biochemical fractionation and candidate gene approaches have been performed to identify the enzymes that execute each step of the related processes (Bernstein, 2001).

The enzyme complex RISC, an effector nuclease for RNAi [Hammond, 2000: see Characterization of RISC (RNA-induced silencing complex) in Drosophila] was isolated from Drosophila S2 cells in which RNAi had been initiated in vivo by transfection with double-stranded RNA (dsRNA). First, an investigation was carried out to discover whether the RISC enzyme, and the enzyme that initiates RNAi through processing of dsRNA into 22-nucleotide sequences, are distinct activities. RISC activity can be largely cleared from extracts by high-speed centrifugation (100,000g for 60 min), whereas the activity that produces 22-nucleotide sequences remains in the supernatant. This simple fractionation indicates that RISC and the 22-nucleotide sequence-generating activity may be separable. However, it seems probable that these enzymes interact at some point during the silencing process, and it remains possible that initiator and effector enzymes share common subunits (Bernstein, 2001).

RNase III family members are among the few nucleases that show specificity for dsRNA. Analysis of the Drosophila and Caenorhabditis elegans genomes revealed several types of RNase III enzymes: (1) the canonical RNase III, which contains a single RNase III signature motif and a dsRNA-binding domain (dsRBD); (2) a class represented by Drosha, a Drosophila enzyme that contains two RNase III motifs and a dsRBD (CeDrosha in C. elegans); (3) a class that contains two RNase III signatures and an amino-terminal helicase domain (for example, Drosophila CG4792 (Dicer 1) and CG6493 (Dicer 2) ; C. elegans K12H4.8), which have been proposed as potential RNAi nucleases. Representatives of all three classes were tested for the ability to produce discrete RNAs of ~22 nucleotides from dsRNA substrates (Bernstein, 2001).

To test the dual RNase III enzymes, variants of Drosha and CG4792 (subsequently called Dicer-1) tagged with the T7 epitope were prepared. These were expressed in transfected S2 cells and isolated by immunoprecipitation using antibody-agarose conjugates. Treatment of the dsRNA with the CG4792 immunoprecipitate yielded fragments of about 22 nucleotides, similar to those produced in either the S2 or embryo extracts. Neither the activity in extract nor that in immunoprecipitates depended on the sequence of the RNA substrate, since dsRNAs derived from several genes were processed equivalently. Negative results were obtained with Drosha and with immunoprecipitates of a DExH box helicase (Homeless). Western blotting confirmed that each of the tagged proteins was expressed and immunoprecipitated similarly. Thus, it is concluded that CG4792 may carry out the initiation step of RNAi by producing guide sequences of about 22 nucleotides from dsRNAs (Bernstein, 2001).

An antiserum directed against the carboxy terminus of the Dicer protein immunoprecipitates a nuclease activity (from either the Drosophila embryo extracts or from S2 cell lysates) that produces RNAs of about 22 nucleotides from dsRNA substrates. The putative guide RNAs that are produced by the Dicer enzyme precisely co-migrate with 22-nucleotide sequences that are produced in extract, and with 22-nucleotide sequences that are associated with the RISC enzyme. The enzyme that produces guide RNAs in Drosophila embryo extracts is ATP dependent. Depletion of ATP results in a roughly sixfold reduction of dsRNA cleavage rate and in the production of RNAs with a slightly lower mobility. Of note, both Dicer immunoprecipitates and extracts from S2 cells require ATP for the production of ~22-nucleotide sequences. The accumulation of lower-mobility products was not observed in these cases, although ~22-nucleotide sequences were routinely observe in ATP-depleted embryo extracts. The requirement of Dicer nuclease for ATP is an unusual property, and may indicate that unwinding of guide RNAs by the helicase domain is required for the enzyme to act catalytically (Bernstein, 2001).

For efficient induction of RNAi in C. elegans and in Drosophila, the initiating RNA must be double-stranded and must also be several hundred nucleotides in length. Similarly, Dicer is inactive against single-stranded RNAs regardless of length. The enzyme can digest both 200- and 500-nucleotide dsRNAs, but is significantly less active with shorter substrates. In contrast, Escherichia coli RNase III can digest to completion dsRNAs of either 35 or 22 nucleotides. This suggests that the substrate preferences of the Dicer enzyme may contribute to, but not wholly determine, the size dependence of RNAi (Bernstein, 2001).

These results indicate that the process of RNAi can be divided into at least two distinct steps. Initiation of RNAi would occur upon processing of a dsRNA by Dicer into ~22-nucleotide guide sequences, although the possibility that another Dicer-associated nuclease may participate in this process cannot be formally excluded. These guide RNAs would be incorporated into a distinct nuclease complex (RISC) that targets single-stranded mRNAs for degradation. An implication of this model is that the guide sequences are themselves derived directly from the dsRNA that triggers the response. In accord with this model, it has been shown that 32P-labelled, exogenous dsRNAs that have been introduced into S2 cells by transfection are incorporated into the RISC enzyme as 22-nuclotide sequences (Bernstein, 2001).

With the identification of Dicer as a potential catalyst of the initiation step of RNAi, the biochemical basis of this unusual mechanism of gene regulation is now open to investigation. It is now important to determine whether the conserved family members from other organisms, particularly mammals, also have a function in dsRNA-mediated gene regulation (Bernstein, 2001).

Dicer-1-dependent Dacapo suppression acts downstream of Insulin receptor in regulating cell division of Drosophila germline stem cells

It is important to understand the regulation of stem cell division because defects in this process can cause altered tissue homeostasis or cancer. The cyclin-dependent kinase inhibitor Dacapo (Dap), a p21/p27 homolog, acts downstream of the microRNA (miRNA) pathway to regulate the cell cycle in Drosophila germline stem cells (GSCs). Tissue-extrinsic signals, including insulin, also regulate cell division of GSCs. Intrinsic and extrinsic regulators intersect in GSC division control; the Insulin receptor (InR) pathway regulates Dap levels through miRNAs, thereby controlling GSC division. Using GFP-dap 3'UTR sensors in vivo, this study shows that in GSCs the dap 3'UTR is responsive to Dicer-1, an RNA endonuclease III required for miRNA processing. Furthermore, the dap 3'UTR can be directly targeted by miR-7, miR-278 and miR-309 in luciferase assays. Consistent with this, miR-278 and miR-7 mutant GSCs are partially defective in GSC division and show abnormal cell cycle marker expression, respectively. These data suggest that the GSC cell cycle is regulated via the dap 3'UTR by multiple miRNAs. Furthermore, the GFP-dap 3'UTR sensors respond to InR but not to TGF-beta signaling, suggesting that InR signaling utilizes Dap for GSC cell cycle regulation. The miRNA-based Dap regulation may act downstream of InR signaling; Dcr-1 and Dap are required for nutrition-dependent cell cycle regulation in GSCs and reduction of dap partially rescues the cell cycle defect of InR-deficient GSCs. These data suggest that miRNA- and Dap-based cell cycle regulation in GSCs can be controlled by InR signaling (Yu, 2009).

Previous studies have shown that miRNAs may regulate Dap, thereby controlling the cell division of GSCs. This study shows that the dap 3'UTR directly responds to miRNA activities in GSCs. Using luciferase assays, miR-7, miR-278 and miR-309 were identified as miRNAs that can directly repress Dap through the dap 3'UTR in vitro. Although miR-278 and miR-7 play a role in regulating GSC division and cell cycle marker expression, respectively, neither of these mutants showed as dramatic a defect in the GSC cell cycle as Dcr-1-deficient GSCs. Thus, the dap 3'UTR may serve to integrate the effect of multiple miRNAs during cell cycle regulation. It remains possible that some miRNAs involved in this process remain to be identified. It was further shown that InR signaling controls the dap 3'UTR in GSCs. This led to an exploration of the interaction between InR signaling and miRNA/Dap cell cycle regulation. GSCs deficient for InR or Dcr-1 show similar cell cycle defects. Using starvation to control InR signaling, it was shown that both Dcr-1 and dap are required for proper InR signaling-dependent regulation of GSC division. Further, reduction of dap partially rescues the cell division defect of the InR mutant GSCs, suggesting that InR signaling regulates the cell cycle via Dap. These results suggest that miRNAs and Dap act downstream of InR signaling to regulate GSC division (Yu, 2009).

The data suggest that multiple miRNAs can regulate the 3'UTR of dap: miR-7, miR-278 and miR-309 can regulate the dap 3'UTR directly, whereas bantam and miR-8 may regulate it indirectly, or through cryptic MREs in the dap 3'UTR. Using GFP sensor assays, it was also shown that the dap 3'UTR may be directly regulated by miRNAs in the GSCs in vivo. However, which specific miRNAs control endogenous Dap levels in Drosophila GSCs remains unknown. Mammalian p21cip1 has also been shown to be a direct target for specific miRNAs of the miR-106 family, including miR-290s and miR-372. Further, the mouse miR-290 family has recently been identified as regulating the G1-S transition. In addition, miR-221 and miR-222 have been shown to regulate p27kip1, thereby promoting cell division in different mammalian cancer cell lines. Neither the miR-290 nor miR-220 family is conserved in Drosophila. Together, these results indicate that the CKIs (Dap) might be a common target for miRNAs in regulating the cell cycle in stem cells. However, the specific miRNAs that regulate the CKIs might vary between organisms (Yu, 2009).

This study reveals novel regulatory roles for miR-7 and miR-278 in the GSC cell cycle. miR-7 and miR-278 can directly target Dap. GSCs deficient for miR-278 show a mild but significant reduction in cell proliferation. Ectopic expression of miR-7 in follicle cells reduces the proportion of cells that stain positive for Dap. Furthermore, ablation of miR-7 in GSCs results in a perturbation of the frequency of CycE-positive GSCs. However, the cell division kinetics of miR-7 mutant GSCs is not reduced, by contrast with the dramatic reduction of cell division in Dcr-1-deficient GSCs. It is plausible that miR-7 and miR-278 act in concert with other miRNAs to regulate the level of Dap in GSCs and thereby contribute to cell cycle control in GSCs (Yu, 2009).

The interaction of multiple miRNAs with the dap 3'UTR might integrate information from multiple pathways. Further studies will reveal what regulates miR-7 and miR-278 expression in GSCs and which other miRNAs might act together in Dap regulation. It is known that miR-7 and the transcriptional repressor Yan mutually repress one another in the eye imaginal disk. In this model, Yan prevents transcription of miR-7 until Erk in the Egfr pathway downregulates Yan activity by phosphorylation, thereby permitting expression of miR-7. Conversely, miR-7 can repress the translation of Yan. Thus, a single pulse of Egfr signaling results in stable expression of miR-7 and repression of Yan. Whether similar regulation will be observed between miR-7 and the signaling pathways that regulate GSC division remains to be seen. It has been suggested that miR-7 might regulate downstream targets of Notch, such as Enhancer of split and Bearded. Thus, miR-7 may have a mild repressive effect on multiple targets in GSCs. Further experiments might illuminate this possibility (Yu, 2009).

miR-278, on the other hand, has been implicated in tissue growth and InR signaling. Overexpression of miR-278 promotes tissue growth in eye and wing imaginal discs. Deficiency of miR-278 leads to a reduced fat body, which is similar to the effect of impaired InR signaling in adipose tissue. Interestingly, miR-278 mutants have elevated insulin/Dilp production and a reduction of insulin sensitivity. Furthermore, miR-278 regulates expanded, which may modulate growth factor signaling including InR. Since InR signaling plays important roles in tissue growth and cell cycle control, it will be interesting to further test how miR-278 may regulate InR signaling, and whether InR signaling might regulate miR-278 in a feedback loop in GSCs (Yu, 2009).

Other miRNAs or miRNA-dependent mechanisms might also play roles in Drosophila GSCs. For example, the miRNA bantam is required for GSC maintenance. A recent study has shown that the Trim-NHL-containing protein Mei-P26, which belongs to the same family as Brain tumor (Brat), affects bantam levels and restricts cell growth and proliferation in the GSC lineage (Neumuller, 2008). Interestingly, most miRNAs are upregulated in mei-P26 mutant flies. By contrast, overexpression mei-P26 in bag of marbles (bam) mutants broadly reduces miRNA levels. This suggests that Mei-P26 regulates proliferation and maintenance of GSC lineages via miRNA levels. Since InR signaling cell-autonomously regulates GSC division but not maintenance, the possible interaction between Mei-P26 and InR signaling might be complex (Yu, 2009).

The systemic compensatory effect of insulin secretion in mammals with defective InR signaling is well documented. Insulin levels in mice with liver-specific InR (Insr - Mouse Genome Informatics) knockout are ~20-fold higher than those of control animals owing to the compensatory response of the pancreatic β-cells and impairment of insulin clearance by the liver. Knockout of the neuronal InR also leads to a mild hyperinsulinemia, indicating whole-body insulin resistance. Furthermore, the knockout of components in the InR signaling pathway, such as Akt2 and the regulatory and catalytic subunits of PI3 kinase, also leads to hyperinsulinemia and glucose intolerance. Therefore, a systemic decrease in InR signaling may lead to compensatory responses (Yu, 2009).

To understand the roles of InR signaling in the GSCs while avoiding any systemic compensatory effect the phenotypes of GSC clones were analyzed. Using a panel of cell cycle markers, it was found that InR mutant GSCs show cell cycle defects similar to those of Dcr-1 mutant GSCs: a reduction of cell division rate, an increased frequency of cells staining positive for Dap and CycE, and a decreased frequency of cells staining positive for CycB. Using GFP-dap 3'UTR sensors, it was shown that the dap 3'UTR responds to InR signaling in GSCs, suggesting that InR signaling can regulate Dap expression through the dap 3'UTR. This, together with genetic data indicating that InR/starvation-dependent cell cycle regulation requires Dcr-1 and dap, has led to the proposalthat InR signaling regulates the cell cycle through miRNAs that further regulate Dap levels. Since a reduction in dap only partially rescues the cell cycle defects of InR mutant GSCs, it is possible that InR signaling might also regulate GSC division by additional mechanisms (Yu, 2009).

InR signaling regulates the cell cycle through multiple mechanisms, mainly through the G1-S, but also partly through the G2-M, transition. Recent work has shown a delay in the G2-M transition in GSCs during C. elegans dauer formation. Starvation and InR deficiency may also affect the G2-M checkpoint in Drosophila GSCs (Hsu, 2008). This study has dissected one possible molecular pathway that InR signaling utilizes to regulate the Drosophila GSC G1-S transition and show that InR signaling can control the cell cycle through miRNA-based regulation of Dap (Yu, 2009).

Many studies have connected InR and CKIs to Tor (Target of rapamycin) or Foxo pathways downstream of InR signaling. In S. cerevisiae, the yeast homolog of p21/p27 is upregulated when Tor signaling is inhibited. Foxo, a transcription factor that can be repressed by InR signaling, is known to play important roles in nutrition-dependent cell cycle regulation by upregulating p21 and p27. In C. elegans, starvation causes L1 cell cycle arrest mediated by InR (daf-2) and Foxo (daf-16): InR represses the function of Foxo, thereby downregulating the CKI (cki-1) and upregulating the miRNA lin-4. This study has shown that a miRNA-based regulation of Dap can be coordinated by InR in Drosophila GSCs (Yu, 2009).

Insulin and insulin-like growth factors (Igf1 and Igf2) are known to play important roles in regulating metabolic and developmental processes in many stem cells. In mammals, Igf signaling is required by different stem cell types, including human and mouse ES cells for survival and self-renewal, neural stem cells for expediting the G1-S transition and cell cycle re-entry, and skeletal muscle satellite cells for promoting the G1-S transition via p27kip1 downregulation. This study has dissected the molecular mechanism of the InR pathway in another adult stem cell type, tDrosophila GSCs, showing that InR signaling can regulate stem cell division through miRNA-based downregulation of the G1-S inhibitor Dap. Further studies will reveal whether miRNAs also mediate InR signaling in other stem cell types (Yu, 2009).


PROTEIN STRUCTURE

Amino Acids - 2249

Structural Domains

Dicer contains two RNase III signatures and an amino-terminal helicase domain. A notable feature of the Dicer family is its evolutionary conservation. Homologs are found in C. elegans (K12H4.8), Arabidopsis (for example, CARPEL FACTORY, T25K16.4 and AC012328_1), mammals (Helicase-MOI) and Schizosaccharomyces pombe (YC9A_SCHPO). In fact, the human Dicer family member is capable of generating ~22-nucleotide RNAs from dsRNA substrates: this indicates that these structurally similar proteins may all share similar biochemical functions. Exogenous dsRNAs can affect gene function in early mouse embryos, and these results suggest that this regulation may be accomplished by evolutionarily conserved RNAi machinery (Bernstein, 2001).

In addition to RNase III and helicase motifs, searches of the PFAM database indicate that each Dicer family member also contains a PAZ domain. This sequence was defined on the basis of its conservation in the Zwille/ARGONAUTE/Piwi family that has been implicated in RNAi by mutations in C. elegans (Rde-1) and Neurospora (Qde-2). Although the function of this domain is unknown, it is notable that this region of homology is restricted to two gene families that participate in dsRNA-dependent silencing. Both the ARGONAUTE and Dicer families have also been implicated in common biological processes, namely the determination of stem-cell fates. A hypomorphic allele of carpel factory, a member of the Dicer family in Arabidopsis, is characterized by increased proliferation in floral meristems. This phenotype and a number of other characteristic features are also shared by Arabidopsis ARGONAUTE (ago1-1) mutants. These genetic analyses provide evidence that RNAi may be more than a defensive response to unusual RNAs, but may also have integral functions in the regulation of endogenous genes (Bernstein, 2001).


Dicer-1: Evolutionary Homologs | Regulation & Characterization of RNAi Process | Developmental Biology | Effects of RNAi Depletion | References

date revised: 5 June 2002

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