The RNase III family of double-stranded RNA-specific endonucleases is characterized by the presence of a highly conserved 9 amino acid stretch in their catalytic center known as the RNase III signature motif. The drosha gene, encoding a new member of this family, was isolated in Drosophila melanogaster. Characterization of this gene revealed the presence of two RNase III signature motifs in its sequence that may indicate that it is capable of forming an active catalytic center as a monomer. The Drosha protein also contains an 825 amino acid N-terminus with an unknown function. A search for the known homologues of the Drosha protein revealed that it has a similarity to two adjacent annotated genes identified during C. elegans genome sequencing. Analysis of the genomic region of these genes by the Fgenesh program and sequencing of the EST cDNA clone derived from it revealed that this region encodes only one gene. This newly identified gene in nematode genome shares a high similarity to Drosophila Drosha throughout its entire protein sequence. A potential Drosha homologue is also found among the deposited human cDNA sequences. A comparison of these Drosha proteins to other members of the RNase III family indicates that they form a new group of proteins within this family (Filippov, 2000).
Hundreds of small RNAs of approximately 22 nucleotides, collectively named microRNAs (miRNAs), have been discovered recently in animals and plants. Although their functions are being unravelled, their mechanism of biogenesis remains poorly understood. miRNAs are transcribed as long primary transcripts (pri-miRNAs) whose maturation occurs through sequential processing events: the nuclear processing of the pri-miRNAs into stem-loop precursors of approximately 70 nucleotides (pre-miRNAs), and the cytoplasmic processing of pre-miRNAs into mature miRNAs. Dicer, a member of the RNase III superfamily of bidentate nucleases, mediates the latter step, whereas the processing enzyme for the former step is unknown. Another RNase III, human Drosha, has been identified as the core nuclease that executes the initiation step of miRNA processing in the nucleus. Immunopurified Drosha cleaved pri-miRNA to release pre-miRNA in vitro. Furthermore, RNA interference of Drosha resulted in the strong accumulation of pri-miRNA and the reduction of pre-miRNA and mature miRNA in vivo. Thus, the two RNase III proteins, Drosha and Dicer, may collaborate in the stepwise processing of miRNAs, and have key roles in miRNA-mediated gene regulation in processes such as development and differentiation (Lee, 2003).
MicroRNAs (miRNAs) represent a family of small noncoding RNAs that are found in plants and animals. miRNAs are expressed in a developmentally and tissue-specific manner and regulate the translational efficiency and stability of partial or fully sequence-complementary mRNAs. miRNAs are excised in a stepwise process from double-stranded RNA precursors that are embedded in long RNA polymerase II primary transcripts (pri-miRNA). Drosha RNase III catalyzes the first excision event, the release in the nucleus of a hairpin RNA (pre-miRNA), which is followed by export of the pre-miRNA to the cytoplasm and further processing by Dicer to mature miRNAs. The human DGCR8, the DiGeorge syndrome critical region gene 8, and its Drosophila melanogaster homolog have been characterized. Biochemical and cell-based readouts are provided to demonstrate the requirement of DGCR8 for the maturation of miRNA primary transcripts. RNAi knockdown experiments of fly and human DGCR8 results in accumulation of pri-miRNAs and reduction of pre-miRNAs and mature miRNAs. These results suggest that DGCR8 and Drosha interact in human cells and reside in a functional pri-miRNA processing complex (Landthaler, 2004).
MicroRNAs (miRNAs) are a growing family of small non-protein-coding regulatory genes that regulate the expression of homologous target-gene transcripts. They have been implicated in the control of cell death and proliferation in flies, haematopoietic lineage differentiation in mammals, neuronal patterning in nematodes and leaf and flower development in plants. miRNAs are processed by the RNA-mediated interference machinery. Drosha is an RNase III enzyme that has been implicated in miRNA processing. Human Drosha is a component of two multi-protein complexes. The larger complex contains multiple classes of RNA-associated proteins including RNA helicases, proteins that bind double-stranded RNA, novel heterogeneous nuclear ribonucleoproteins and the Ewing's sarcoma family of proteins. The smaller complex is composed of Drosha and the double-stranded-RNA-binding protein, DGCR8, the product of a gene deleted in DiGeorge syndrome. In vivo knock-down and in vitro reconstitution studies have revealed that both components of this smaller complex, termed Microprocessor, are necessary and sufficient in mediating the genesis of miRNAs from the primary miRNA transcript (Gregory, 2004).
Mature microRNAs (miRNAs) are generated via a two-step processing pathway to yield approximately 22-nucleotide small RNAs that regulate gene expression at the post-transcriptional level. Initial cleavage is catalysed by Drosha, a nuclease of the RNase III family, which acts on primary miRNA transcripts (pri-miRNAs) in the nucleus. Here it is shown that Drosha exists in a multiprotein complex, the Microprocessor, and begin the process of deconstructing that complex into its constituent components. Along with Drosha, the Microprocessor also contains Pasha (partner of Drosha), a double-stranded RNA binding protein. Suppression of Pasha expression in Drosophila cells or Caenorhabditis elegans interferes with pri-miRNA processing, leading to an accumulation of pri-miRNAs and a reduction in mature miRNAs. Finally, depletion or mutation of pash-1 in C. elegans causes de-repression of a let-7 reporter and the appearance of phenotypic defects overlapping those observed upon examination of worms with lesions in Dicer (dcr-1) or Drosha (drsh-1). Considered together, these results indicate a role for Pasha in miRNA maturation and miRNA-mediated gene regulation (Denli, 2004).
A critical step during human microRNA maturation is the processing of the primary microRNA transcript by the nuclear RNaseIII enzyme Drosha to generate the 60-nucleotide precursor microRNA hairpin. How Drosha recognizes primary RNA substrates and selects its cleavage sites has remained a mystery, especially given that the known targets for Drosha processing show no discernable sequence homology. Human Drosha selectively cleaves RNA hairpins bearing a large (10 nucleotides) terminal loop. From the junction of the loop and the adjacent stem, Drosha then cleaves approximately two helical RNA turns into the stem to produce the precursor microRNA. Beyond the precursor microRNA cleavage sites, approximately one helix turn of stem extension is also essential for efficient processing. While the sites of Drosha cleavage are determined largely by the distance from the terminal loop, variations in stem structure and sequence around the cleavage site can fine-tune the actual cleavage sites chosen (Zeng, 2005).
The short integument (sin1) mutation causes a female-specific infertility, and a defect in the control of time to flowering in Arabidopsis. Female sterility of Sin minus plants is due to abnormal ovule integument development and aberrant differentiation of the megagametophyte in a subset of ovules. An additional defect of sin1 mutants is the production of an increased number of vegetative leaf and inflorescence primordia leading to delayed flowering. The delayed flowering phenotype of sin1-1 is not due to a defect in the perception of day length periodicity or in gibberellic acid metabolism. Phenotypes of double mutant combinations of sin1 with terminalflower (tfl1) indicate that SIN1 activity is required for precocious floral induction typical in a tfl1 mutant. Unexpectedly, sin1-1 tfl1-1 plants do not make pollen, thus revealing a novel role for TFL1 in the anther. Early flowers of sin1-1 ap1-1 double mutants are transformed to long inflorescence-like shoots. A genetic model for the role of SIN1 in flowering time control is proposed (A. Ray, 1996).
Maternal gene products deposited in an animal egg determine the polarity of embryonic axes and regulate embryonic cell-cell communication important for morphogenesis. The first maternal-effect embryo-defective mutation discovered in a plant is reported in this study. Recessive mutations in the SHORT INTEGUMENT (SIN1) gene in Arabidopsis were previously shown to influence ovule development and flowering time. A sin1 mutation has a pronounced maternal effect on zygotic embryo development. A homozygous sin1 mutant embryo is normal when nursed by a sin1/+ heterozygous maternal sporophyte. Strikingly, a sin1 or a sin1/+ embryo that is nursed by a sin1 homozygous maternal sporophyte develops morphogenetic defects in the apical-basal and radial axes. The defects resemble those seen in some zygotic-effect embryonic pattern formation mutants. These results imply that in maternal cells the SIN1 gene either codes for or controls the production of a diffusible morphogen necessary for proper zygotic embryogenesis (S. Ray, 1996).
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, because 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 (Jacobsen, 1999).
In metazoans, microRNAs, or miRNAs, constitute a growing family of small regulatory RNAs that are usually 19–25 nucleotides in length. They are processed from longer precursor RNAs that fold into stem-loop structures by the ribonuclease Dicer and are thought to regulate gene expression by base pairing with RNAs of protein-coding genes. In Arabidopsis thaliana, mutations in CARPEL FACTORY (CAF), a Dicer homolog, and those in a novel gene, HEN1, result in similar, multifaceted developmental defects, suggesting a similar function of the two genes, possibly in miRNA metabolism. To investigate the potential functions of CAF and HEN1 in miRNA metabolism, attempts were made to isolate miRNAs from Arabidopsis and examine their accumulation during plant development in wild-type plants and in hen1-1 and caf-1 mutant plants. Eleven miRNAs were isolated, some of which have potential homologs in tobacco, rice, and maize. The putative precursors of these miRNAs have the capacity to form stable stem-loop structures. The accumulation of these miRNAs appears to be spatially or temporally controlled in plant development, and their abundance is greatly reduced in caf-1 and hen1-1 mutants. HEN1 homologs are found in bacterial, fungal, and metazoan genomes. It is concluded that miRNAs are present in both plant and animal kingdoms. An evolutionarily conserved mechanism involving a protein, known as Dicer in animals and CAF in Arabidopsis, operates in miRNA metabolism. HEN1 is a new player in miRNA accumulation in Arabidopsis, and HEN1 homologs in metazoans may have a similar function. The developmental defects associated with caf-1 and hen1-1 mutations and the patterns of miRNA accumulation suggest that miRNAs play fundamental roles in plant development (Park, 2002).
Formation of microRNA (miRNA) requires an RNaseIII domain-containing protein, termed Dicer-1 in animals and Dicer-like1 (Dcl1) in plants, to catalyze processing of an RNA precursor with a fold-back structure. Loss-of-function dcl1 mutants of Arabidopsis have low levels of miRNA and exhibit a range of developmental phenotypes in vegetative, reproductive, and embryonic tissues. Dcl1 mRNA occurs in multiple forms, including truncated molecules that result from aberrant pre-mRNA processing. Both full-length and truncated forms accumulate to relatively low levels in plants containing a functional Dcl1 gene. However, in dcl1 mutant plants, dcl1 RNA forms accumulated to levels several-fold higher than those in Dcl1 plants. Elevated levels of Dcl1 RNAs are also detected in miRNA-defective hen1 mutant plants and in plants expressing a virus-encoded suppressor of RNA silencing (P1/HC-Pro), which inhibits miRNA-guided degradation of target mRNAs. A miRNA (miR162) target sequence was predicted near the middle of Dcl1 mRNA, and a Dcl1-derived RNA with the properties of a miR162-guided cleavage product was identified and mapped. These results indicate that Dcl1 mRNA is subject to negative feedback regulation through the activity of a miRNA (Xie, 2003).
The Schizosaccharomyces pombe genome encodes only one of each of the three major classes of proteins implicated in RNA silencing: Dicer (Dcr1), RNA-dependent RNA polymerase (RdRP; Rdp1), and Argonaute (Ago1). These three proteins are required for silencing at centromeres and for the initiation of transcriptionally silent heterochromatin at the mating-type locus. The introduction of a double-stranded RNA (dsRNA) hairpin corresponding to a green fluorescent protein (GFP) transgene triggers classical RNA interference (RNAi) in S. pombe. That is, GFP silencing triggered by dsRNA reflects a change in the steady-state concentration of GFP mRNA, but not in the rate of GFP transcription. RNAi in S. pombe requires dcr1, rdp1, and ago1, but does not require chp1, tas3, or swi6, genes required for transcriptional silencing. Thus, the RNAi machinery in S. pombe can direct both transcriptional and posttranscriptional silencing using a single Dicer, RdRP, and Argonaute protein. These findings suggest that these three proteins fulfill a common biochemical function in distinct siRNA-directed silencing pathways (Sigova, 2004).
This study demonstrates that a dsRNA derived from a hairpin transcript can trigger posttranscriptional silencing of a corresponding mRNA in S. pombe. A similar hairpin transcript, corresponding to the ura4 locus has also been shown (Schramke, 2003) to trigger transcriptional silencing. In both studies, silencing triggered by a hairpin transcript require the RNAi machinery -- Dcr1, Rdp1, and Ago1. Transcriptional silencing, unlike posttranscriptional silencing, requires components of the transcriptional silencing apparatus: Chp1, Tas3, or Swi6. Robust silencing by both pathways requires the chromodomain protein Clr4, which appears to play a role in siRNA biogenesis or stability. Why does the GFP hairpin construct presented in this study trigger exclusively posttranscriptional silencing, whereas the previously studied ura4 hairpin triggered transcriptional silencing? One possible explanation is that the GFP hairpin used here includes an efficiently spliced intron between the two arms of the hairpin. It is presumed that splicing of the intron promotes the accumulation of GFP dsRNA in the cytoplasm. In contrast, the ura4 hairpin construct of Schramke (2003) contains an unspliced spacer sequence between the hairpin arms. Thus, the ura4 hairpin may be localized largely to the nucleus. A difference in subcellular localization might explain the different results obtained by the two studies. Alternatively, silencing of ura4 by the ura4-specific hairpin might comprise a mixture of transcriptional and posttranscriptional silencing. In this case, transcriptional silencing might not occur at the adh1 locus, even if the GFP hairpin-derived siRNAs trigger histone modification, perhaps because the gene is strongly expressed or is in a region of the genome otherwise refractory to heterochromatin formation. Nonetheless, the current data, together with those of Schramke (2003), clearly show that at least two distinct silencing responses can be initiated by a common RNAi machinery, without resorting to specialized forms of Dicer, RdRP, or Argonaute proteins. The demonstration that fission yeast contain a functional RNAi pathway now provides a simplified, genetically tractable model in which to study how the nature of the silencing trigger or of the silencing target determines the silencing pathway evoked -- posttranscriptional or transcriptional (Sigova, 2004).
RNA silencing phenomena, known as post-transcriptional gene silencing in plants, quelling in fungi, and RNA interference (RNAi) in animals, are mediated by double-stranded RNA (dsRNA) and mechanistically intersect at the ribonuclease Dicer. The 218 kDa human Dicer has been cloned: its ribonuclease activity and dsRNA-binding properties have been characterized. The recombinant enzyme generates approximately 21-23 nucleotide products from dsRNA. Processing of the microRNA let-7 precursor by Dicer produces an apparently mature let-7 RNA. Mg(2+) was required for dsRNase activity, but not for dsRNA binding, thereby uncoupling these reaction steps. ATP is dispensable for dsRNase activity in vitro. The Dicer.dsRNA complex formed at high KCl concentrations is catalytically inactive, suggesting that ionic interactions are involved in dsRNA cleavage. The putative dsRNA-binding domain located at the C-terminus of Dicer binds dsRNA in vitro. Human Dicer expressed in mammalian cells colocalizes with calreticulin, a resident protein of the endoplasmic reticulum. Availability of the recombinant Dicer protein will help improve understanding of RNA silencing and other Dicer-related processes (Provost, 2002).
In animals, the double-stranded RNA-specific endonuclease Dicer produces two classes of functionally distinct, tiny RNAs: microRNAs (miRNAs) and small interfering RNAs (siRNAs). miRNAs regulate mRNA translation, whereas siRNAs direct RNA destruction via the RNA interference (RNAi) pathway. In human cell extracts, the miRNA let-7 naturally enters the RNAi pathway, which suggests that only the degree of complementarity between a miRNA and its RNA target determines its function. Human let-7 is a component of a previously identified, miRNA-containing ribonucleoprotein particle, which is an RNAi enzyme complex. Each let-7-containing complex directs multiple rounds of RNA cleavage, which explains the remarkable efficiency of the RNAi pathway in human cells (Hutvagner, 2002).
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, the precise 5' and 3' ends of the let-7 precursors in human cells have been characterized. They harbor a 5'-phosphate and a 3'-OH and 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, these 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 (Basyuk, 2003)
Dicer is a multidomain ribonuclease that processes double-stranded RNAs (dsRNAs) to 21 nt small interfering RNAs (siRNAs) during RNA interference, and excises microRNAs from precursor hairpins. Dicer contains two domains related to the bacterial dsRNA-specific endonuclease, RNase III, which is known to function as a homodimer. Based on an X-ray structure of the Aquifex aeolicus RNase III, models of the enzyme interaction with dsRNA, and its cleavage at two composite catalytic centers, have been proposed. Mutations were generated in human Dicer and Escherichia coli RNase III residues implicated in the catalysis, and their effect on RNA processing was studed. The results indicate that both enzymes have only one processing center, containing two RNA cleavage sites and generating products with 2 nt 3' overhangs. Based on these and other data, it is proposed that Dicer functions through intramolecular dimerization of its two RNase III domains, assisted by the flanking RNA binding domains, PAZ and dsRBD (Zhang, 2004).
To obtain direct evidence that Dicer is involved in gene silencing, the effectiveness of RNAi in a C. elegansstrain containing a null mutation in the Dicer homolog (dcr-1), was examined. DCR-1 is encoded by an 8165-base pair (bp) gene in C. elegans and contains an NH2-terminal DExH/DEAD-box type RNA helicase domain, two RNase III-like domains, and a COOH-terminal dsRNA binding motif. Animals with a deletion in dcr-1 that removes a 2470-bp fragment spanning a region from exon 13 to intron 18 were obtained from the C. elegans gene knockout consortium. The deletion removes the NH2-terminal portion of the first RNase III domain and is also predicted to introduce multiple stop codons into the reading frame. dcr-1(-/-) animals are sterile, suggesting that DCR-1 has an essential role in vivo and also emphasizing that the deletion creates a loss-of-function allele (Knight, 2001).
In C. elegans, RNAi is typically initiated by injecting or feeding dsRNA, and gene silencing is subsequently observed in the F1 progeny. Because dcr-1(-/-) animals are sterile and do not give rise to progeny, a transgenic line was used in which RNAi could be monitored in individual animals, without waiting for subsequent generations. The line was constructed by microinjecting DNA encoding green fluorescent protein (GFP) (sur5::GFP), as well as a previously described vector containing an RNA hairpin matching the GFP sequence, under the control of a heat shock promoter [hsp16-2pGFP(IR)]. In the transgenic line isolated, heat shock produces an easily discernable RNAi phenotype in heat-shocked animals, so it is not necessary to analyze progeny. The transgenic line was made with dcr-1(+/-) animals and dcr-1(-/-) progeny were examined for RNAi resistance after heat shock. Whereas wild-type animals exhibit robust RNAi measured by a loss in GFP fluorescence, animals homozygous for the dcr-1 deletion are RNAi defective and continue to exhibit a strong fluorescence. These results are consistent with the idea that dcr-1 is required for RNAi (Knight, 2001).
Gene silencing by RNAi is known to involve the degradation of the targeted mRNA. To obtain molecular evidence that dcr-1 is required for RNAi, as well as to monitor the effects of the dcr-1 deletion on RNAi of other genes, semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure mRNA levels after RNAi. When dsRNA corresponding to the mpk-1 gene was injected into L4 worms, wild-type animals exhibited reduced mpk-1 mRNA levels, whereas mpk-1 mRNA remained abundant in dcr-1(-/-) animals . These results, and similar results with dsRNA to gld-1, again suggest that DCR-1 is required for RNAi. However, when animals were injected with dsRNA to unc-54, decreased levels of unc-54 mRNA were observed in both wild-type and dcr-1(-/-) animals, consistent with a normal RNAi response (Knight, 2001).
In the above experiments, dcr-1(-/-) animals were resistant to RNAi of genes expressed in the germ line (mpk-1 and gld-1) but showed a normal RNAi response for unc-54, a somatic gene. Thus, dcr-1 may be similar to the genes mut-7 and rde-2, or ego-1, which are required for RNAi of germ line expressed genes, but not for somatic genes. Consistent with this idea, it was found that dcr-1 mutants showed a normal RNAi response when dsRNA corresponding to another somatic gene, unc-22, was injected. dcr-1(-/-) animals were also sensitive to RNAi when fed bacteria expressing unc -22 dsRNA (Knight, 2001).
It was also noticed that the requirement of RNAi for the dcr-1 gene depended on the method used to deliver the dsRNA. For example, dcr-1 was required for RNAi of the GFP transgene by the heat shock-inducible RNA hairpin. However, when gfpdsRNA was injected into the dcr-1(-/-) animals carrying the GFP transgene, 100% of dcr-1(-/-) animals had reduced fluorescence (n = 14). The data indicate that dcr-1(-/-) animals are defective for RNAi in some but not all cases. Possibly, gene silencing by dsRNA can occur by multiple pathways, some that require DCR-1 and some that do not. Alternatively, the RNAi defects of dcr-1(-/-) animals may be partially rescued by maternal dicer (mRNA or protein) that persists in the F1 progeny. However, if this is the case, the maternal DCR-1 must not be available, or sufficient, to rescue all RNAi (Knight, 2001).
To begin to understand the role of dcr-1 in germ line development, germ line morphology was compared in wild-type and dcr-1(-/-) adult hermaphrodites using differential interference contrast microscopy and DAPI epifluorescence staining. Normally, in adult wild-type animals, the germ line develops in a defined and largely invariant manner. Moving from the distal region proximally, germ cells proliferate, enter meiosis, and differentiate into oocytes in the loop region and proximal gonad. Oocytes are fertilized as they are pulled through the spermatheca into the uterus. In dcr-1(-/-) animals, no gross defects were observed in chromosome morphology in the distal region of the gonad; however, several defects are seen in the proximal region. Elongated oocytes are found even before the loop of the gonad. Furthermore, as they migrate proximally, oocytes appear misshapen, lack clear delineation, and remain unfertilized. Nuclei in proximal oocytes also appear abnormal, often appearing nonspherical. DAPI staining reveals areas of intense staining in enlarged proximal nuclei suggestive of DNA replication without cell division (endomitosis). Vulval bursting was observed in many, but not all, dcr-1(-/-) animals. The burst vulva phenotype is also observed in animals containing a mutation in let-7 (see Drosophila microRNA encoding gene let-7), which encodes a 21- to 22-nt RNA important for developmental timing. Because the mature let-7 RNA is similar to the size of putative Dicer products and is thought to be processed from a base-paired hairpin, the burst vulva phenotype may indicate that let-7 processing is defective in the dcr-1 mutants. Taken together, the phenotypes of dcr-1(-/-) animals indicate that DCR-1 has multiple and important roles in vivo (Knight, 2001).
RNAi is a gene-silencing phenomenon triggered by double-stranded (ds) RNA and involves the generation of 21 to 26 nt RNA segments that guide mRNA destruction. In Caenorhabditis elegans, lin-4 and let-7 encode small temporal RNAs (stRNAs) of 22 nt that regulate stage-specific development. Inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-1), and two homologs of rde-1 (alg-1 and alg-2), cause heterochronic phenotypes similar to lin-4 and let-7 mutations. dcr-1, alg-1, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7 stRNAs. These findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation (Grishok, 2001).
Genetic studies in C. elegans have identified several genes essential for RNA interference. Probable null mutations in rde-1 (for RNAi defective) cause a complete lack of RNAi but no other discernible phenotypes. rde-1 encodes a 1020 amino acid protein that is a member of a large family of proteins found in a wide range of eukaryotes. Members of the RDE-1 family have two conserved domains of unknown biochemical function. The 300 amino acid PIWI domain located in the C-terminal region of these homologs shows the highest degree of sequence conservation. The 110 amino acid PAZ domain is located N-terminal to the PIWI domain and is also found in the Dicer family of proteins. RDE-1 homologs in the fungus, Neurospora, and the plant, Arabidopsis, have also been implicated in PTGS (post-transcriptional gene silencing) mechanisms suggesting that RDE-1 family members not only share conserved structures but also have conserved functions in gene silencing in three kingdoms of eukaryotic organisms (Grishok, 2001 and references therein).
Mutations in rde-1 homologs have also been shown to have developmental consequences. For example, in Drosophila, the ago1 gene is required for embryogenesis (Kataoka, 2001), the piwi gene is required for the maintenance of the germline stem cell population, and aubergine is required for the proper expression of the germline determinant Oskar (Wilson, 1996). Additionally, aubergine (also known as Sting) has been implicated in the PTGS-like suppression of the repetitive Stellate locus in the Drosophila germline (Schmidt, 1999). In Arabidopsis two very similar genes, argonaute (ago1) and pinhead/zwille, are required for stem cell patterning of the plant meristem. argonaute is also necessary for PTGS in Arabidopsis. The C. elegans genome contains 23 homologs of rde-1 including orthologs of both piwi and ago1. Previous studies have shown that the C. elegans piwi and ago1 orthologs have germline and possibly additional developmental functions. The pleiotropic nature of the defects associated with loss-of-function mutations in members of this family could reflect discrete regulatory functions in numerous developmental events or alternatively might reflect a more general misregulation of silencing mechanisms that are necessary to insure proper stem cell maintenance and differentiation (Grishok, 2001 and references therein).
cDNA clones for 14 rde-1 homologs were tested for developmental functions by RNAi. dsRNAs derived from two closely related genes, F48F7.1 and T07D3.7, which have been named alg-1 and alg-2 (for argonaute like genes), induce developmental phenotypes in the progeny of injected animals, including a tendency to burst at the vulva, and a lack of the adult specific alae, longitudinal stripes that run the length of the cuticle on both sides of the adult animal. In addition these dsRNAs induce incompletely penetrant slow growth and germline abnormalities. The other 12 genes assayed did not exhibit discernable developmental phenotypes (Grishok, 2001).
The alg-1 and alg-2 DNA sequences are 80% identical at the nucleotide level, suggesting a recent duplication of these genes, although they map to distinct chromosomes. This level of similarity is within the range where partial cross-interference is expected in RNAi assays. To target only alg-1 or alg-2, dsRNAs were prepared from short 5' unique segments of each gene. The dsRNA prepared from the unique segment of alg-1 produces the same vulval bursting phenotype, although at a reduced frequency relative to that observed with longer dsRNAs. No RNAi phenotype was observed after injections of the unique segment of alg-2 (Grishok, 2001).
A deletion allele of alg-2 was obtained from the C. elegans gene knockout consortium. This allele, alg-2(ok304), is an out-of-frame deletion that removes the nucleotides encoding amino acids 34-374, including the PAZ domain, and terminates after encoding 8 additional amino acids from reading frame two. It is therefore likely to be a null allele of alg-2. The RNAi experiments suggest that alg-2 may be a nonessential gene, and consistent with this finding the alg-2(ok304) homozygotes are viable and show, at most, subtle defects in fertility and development (Grishok, 2001).
It was next asked if alg-1 and alg-2 might have overlapping functions; dsRNAs prepared from both genes were coinjected and alg-1 dsRNA was injected into alg-2(ok304) homozygotes. Consistent with a shared function, coinjection of alg-1 and alg-2 dsRNAs causes enhanced larval lethality and also induces an embryonic lethal phenotype. Injection of alg-1 dsRNA into alg-2(ok304) homozygous animals results in a fully penetrant embryonic lethal phenotype identical to that observed in the double RNAi experiment. No such synergy was observed when alg-1 dsRNA was injected with dsRNAs prepared from other rde-1 family members. These findings indicate that alg-1 and alg-2 have overlapping functions in both embryogenesis and larval development. Efficient induction of the larval developmental phenotypes require the injection of full-length alg-1 dsRNA, a procedure that appears to partially inhibit alg-2. Therefore, animals produced in such experiments are referred to as 'alg-1/alg-2' RNAi animals (Grishok, 2001).
Finally, alg-1 and alg-2 were assayed for possible roles in RNAi. The alg-2(ok304) homozygotes are fully sensitive to RNAi, and likewise the inhibition of alg-1 or alg-2 by RNAi does not suppress RNAi targeting a second gene. These findings suggest that alg-1 and alg-2 are not necessary for RNAi. Nevertheless, it remains possible that these genes might have some redundant function in RNAi with rde-1 or with other members of this gene family (Grishok, 2001).
The C. elegans gene K12H4.8, which has been named dcr-1, is predicted to encode a protein related to the Drosophila Dicer (Bernstein, 2001) and the Arabidopsis Carpel Factory (Jacobsen, 1999) proteins implicated in RNAi and regulation of development, respectively. A previous study has shown that RNA interference of Drosophila Dicer can induce a partial loss of RNAi (Bernstein, 2001). RNAi of C. elegans dcr-1 was used to assess its role in developmental control and RNA interference. dcr-1(RNAi) induces developmental abnormalities during larval growth that are very similar to those induced by alg-1/alg-2(RNAi). These include a protruding and non-functional vulva, and a tendency to burst at the vulva shortly after the molt from the larval to the adult stage. In addition, dcr-1(RNAi) animals frequently exhibited faint or missing adult-specific alae (Grishok, 2001).
Although the phenotypes induced by dcr-1(RNAi) were similar to those induced by alg-1/alg-2(RNAi), dcr-1(RNAi) phenotypes were less penetrant. For example, 91% of the alg-1/alg-2(RNAi) animals lacked the adult-specific alae while only 19% of the dcr-1(RNAi) animals completely lacked the alae. This finding could indicate that dcr-1 has only a relatively minor role in the specification of the alae; alternatively, it might reflect a difficulty in inhibiting dcr-1 function via RNAi. For example, if dcr-1 is required for RNAi in C. elegans as it appears to be in Drosophila, then the use of RNAi to target dcr-1 may, at best, diminish its activity (Grishok, 2001).
The dcr-1(RNAi) phenotype was compared to the phenotype of animals homozygous for mutations in dcr-1. Three noncomplementing mutant strains were obtained that define the dcr-1 locus. Two of these, let-740(s2624) and let-740(s2795), were identified in an extensive genetic screen for mutations balanced by the free duplication sDp3. The third allele, dcr-1(ok247), was made by the C. elegans gene knockout consortium. The let-740(s2624) and let-740(s2795) mutations result in premature stop codons while dcr-1(ok247) is an out-of-frame deletion allele removing residues 708 through 1321 and terminating after expression of 15 amino acid residues from intronic sequences. All of these lesions are likely to severely disrupt DCR-1 protein expression; the s2624 allele would encode a protein of only 59 amino acids lacking all of the recognizable functional motifs, while the latter two alleles would encode truncated proteins lacking the PAZ, RNase III, and dsRBP domains. All three mutant dcr-1 strains exhibit a similar, fully penetrant, sterile phenotype. Homozygous hermaphrodites produce germ cells, including both sperm and oocytes, but for unknown reasons fail to produce embryos. In addition, all three strains exhibit adult cuticle and vulval defects identical to the defects induced by dcr-1(RNAi), including a protruding vulva and occasional vulval bursting as well as faint or missing alae. Because the let-740 mutations are allelic to dcr-1(ok247), the more descriptive name, dcr-1, will be used to refer to this gene (Grishok, 2001).
The severity of the phenotypes observed in the dcr-1 homozygous mutants is dependent on the maternal genotype, suggesting that dcr-1(+) activity is provided maternally. If dcr-1(+) activity is provided maternally, then RNAi of dcr-1 into a dcr-1 heterozygous mother might be expected to enhance the cuticle defects or cause additional phenotypes in the homozygous mutant progeny of the injected animal. Consistent with this possibility, the homozygous mutant class of progeny from dcr-1 heterozygous mothers injected with dcr-1 dsRNA arrests as embryos at a developmental stage similar to that observed in the double RNAi targeting alg-1 and alg-2. These findings suggest that maternal dcr-1(+) activity rescues essential functions of dcr-1 in the homozygous embryos and larvae and that RNAi of dcr-1 depletes this maternal activity. Because RNAi of dcr-1 efficiently inhibits dcr-1 activities required for larval development without inducing sterility or embryonic lethality, dcr-1(RNAi) was used for the subsequent developmental studies (Grishok, 2001).
Finally, it was asked if homozygous dcr-1 mutants are sensitive to RNAi. The conceptually straightforward experiment of assaying RNAi in the complete absence of dcr-1 is, unfortunately, not feasible since dcr-1 is required for viability of the animal. The best experiments that can be done are to assay for sensitivity to RNAi in animals where dcr-1 activity has been decreased. dcr-1(ok247) homozygous animals were tested for sensitivity to dsRNA delivered by injection into their mother or directly into the homozyous L4 larvae. In both assays nearly normal levels of RNAi were observed. This observation could indicate that maternal dcr-1(+) activity can rescue RNAi in dcr-1 homozygous mutant progeny just as it appears to rescue the developmental and alae defects described above. Consistent with this idea, other RNAi pathway mutants including rde-1 and rde-4 homozygotes are strongly rescued by one maternal dose of rde(+) activity. Because dsRNA targeting dcr-1 induces strong larval developmental defects, it was next asked if dcr-1(RNAi) might sufficiently reduce dcr-1 activity to cause an RNAi-deficient phenotype. For this assay, dcr-1 dsRNA was injected into adult hermaphrodites and then assayed for sensitivity to RNAi targeting a second gene. In experiments targeting two different genes, a significant reduction of RNAi was observed among the progeny of dcr-1(RNAi) animals but not among control animals injected with unrelated dsRNAs. These results support the findings from Bernstein (2001) that implicate Drosophila Dicer in RNAi and suggest that DCR-1 may have a similar activity in C. elegans (Grishok, 2001).
The combination of vulval and adult cuticle maturation defects caused by RNAi of alg-1/alg-2 and dcr-1 is reminiscent of phenotypes resulting from mutations in the genes lin-4 and let-7. The lin-4 and let-7 genes promote transitions from earlier to later cell fates and, thus, mutations in these genes cause reiteration of cell divisions typical of earlier larval stages, a hallmark of genes that regulate developmental timing (such genes have been termed 'heterochronic genes'). For example, loss-of-function mutations in let-7 result in a failure of larval seam cells in the hypodermis to progress to the adult-specific program of terminal differentiation indicated by the production of the adult-specific alae -- instead, the cells repeat the late larval type of divisions. These reiterated divisions contribute to an unstable vulval structure and failure to form a cuticle with adult alae (Grishok, 2001).
The developmental defects in alg-1/alg-2 and dcr-1 RNAi animals also result from temporal misspecifications in the seam cell lineages. To aid in the observation of seam cell divisions, a transgenic strain was utilized that drives GFP expression specifically in the seam cell nuclei. Normally, the ten seam cells present at hatching divide to generate 16 cells during the second larval stage. Although these 16 cells divide at the succeeding third and fourth larval transitions, only one daughter cell maintains the seam cell fate, so that the total number of GFP-expressing seam cells in the adult is 16 (Grishok, 2001).
RNAi of either dcr-1 or alg-1/alg-2 results in adults with extra seam cells that arise from reiterated L2 type divisions. Most progeny of dcr-1 and alg-1/alg-2 dsRNA-injected parents had normal seam cell divisions until the L3 stage, when reiterations of L2 type divisions were common. Many animals showed mixed patterns of stage-specific divisions, a phenotype similar to that observed previously in heterochronic mutants (daf-12, for example). The number of seam cells observed in dcr-1(RNAi) adults ranged from 16 to 33, with an average of 21, and only 15% showed the normal number of 16 seam cells; alg-1/alg-2(RNAi) adults exhibited 18-36 seam cells with an average of 25. The dcr-1 and alg-1/alg-2 (RNAi) progeny also repeated L3 or L4 seam cell division programs into adulthood, when normally these cells would stop dividing and become terminally differentiated (Grishok, 2001).
Inappropriate seam cell division patterns were consistently observed in L3 through later stages in dcr-1(RNAi) and alg-1/alg-2(RNAi) animals. However, because of the likely incomplete RNAi of dcr-1 and the redundancy of alg-1 and alg-2, it was not possible to establish the precise point in larval development where these genes are first required. Additional support that these genes may act earlier in larval development comes from the seam cell division pattern displayed by the more strongly affected animals obtained by coinjecting dsRNAs targeting portions of both alg-1 and alg-2 . In these experiments, reiterations of L1-type divisions were observed, in addition to repetition of later stage patterns (Grishok, 2001).
The similarity of phenotypes described above to those of the heterochronic genes lin-4 and let-7 raised the possibility that alg-1, alg-2, and dcr-1 might act upstream of the lin-4 or let-7 stRNAs or might be necessary for their regulatory activities. The targets of lin-4 and let-7 include the lin-14 and lin-41 mRNAs. Genetic studies suggest that lin-4 and let-7 stRNAs directly regulate lin-14 and lin-41 through complementary sequences in their 3'UTRs. Because the retarded phenotypes of lin-4 and let-7 are caused in part by failure to downregulate their target genes, mutations in lin-14 and lin-41 partially suppress the lin-4 and let-7 mutant phenotypes. To determine if alg-1/alg-2 and dcr-1 RNAi animals exhibit a similar genetic relationship with lin-14 and lin-41 mutants, dsRNA injections were performed in lin-14 and lin-41 mutant backgrounds. Significant suppression of the RNAi-induced alg-1/alg-2 and dcr-1 heterochronic phenotypes was found, including alae and vulval defects, by the lin-14(n179) and lin-41(ma104) nonnull mutations. In addition, the penetrant germline phenotype associated with alg-1/alg-2(RNAi) was partially suppressed by the lin-41 and lin-14 mutations, but the synthetic lethal phenotype associated with double alg-1/alg-2(RNAi) was not suppressed. In control RNAi experiments, the lin-14 and lin-41 mutant strains were fully sensitive to RNAi. These findings are consistent with the idea that the retarded heterochronic phenotypes induced by alg-1/alg-2 and dcr-1(RNAi) are caused, at least in part, by misregulation of lin-14 and lin-41 (Grishok, 2001).
lin-4 and let-7 are expressed as longer, approximately 70 nt RNAs that are predicted to fold into structures containing regions of double-stranded RNA. Because Drosophila Dicer cleaves introduced dsRNAs into fragments of approximately 22 nt (Bernstein, 2001), it was hypothesized that the heterochronic phenotypes caused by dcr-1 (RNAi) may be due to a defect in the processing of the larger, potentially dsRNA, forms of lin-4 and let-7 into the 22 nt stRNAs. To test this idea progeny were collected from mothers subjected to dcr-1(RNAi) and Northern blot analyses were performed to monitor the size and abundance of the lin-4 and let-7 RNAs. Because alg-1/alg-2 (RNAi) causes a similar heterochronic phenotype but acts at an unknown step in the pathway, lin-4 and let-7 processing were also monitored in alg-1/alg-2 (RNAi) animals (Grishok, 2001).
Both dcr-1 and alg-1/alg-2(RNAi) animals exhibited a marked accumulation of the lin-4 long form at both L3-L4 and adult stages. The same RNA preparations from the dcr-1 or alg-1/alg-2 (RNAi) animals were probed for the expression of let-7. It was found that, as with lin-4, let-7 processing depends on dcr-1 activity but, in contrast, does not appear to depend on alg-1/alg-2 activity. lin-4 and let-7 stRNA processing were monitored in dcr-1(ok247) homozygotes and in animals specifically depleted for either alg-1 or alg-2. In this experiment RNAs prepared from each population were simultaneously probed for expression of lin-4 and let-7 RNA. As with dcr-1(RNAi), the ok247 homozygotes exhibit a significant accumulation of both lin-4 and let-7 long forms. A gene-specific dsRNA targeting alg-1 induces accumulation of the pre-lin-4 RNA but not pre-let-7, and similarly, alg-2(ok304) animals exhibits a slight accumulation of pre-lin-4 and little or no accumulation of pre-let-7 (Grishok, 2001).
The quantity of the short forms of the lin-4 and let-7 stRNAs consistently appeared to be reduced in RNA populations prepared from alg-1/alg-2(RNAi), dcr-1(RNAi), and dcr-1(ok247) animals, while control RNA populations prepared from animals undergoing RNAi of the cuticle collagen gene rol-6 exhibited normal levels of lin-4 and let-7 stRNAs. This apparent reduction in let-7 stRNA level was observed even in alg-1/alg-2(RNAi) populations where no significant accumulation of pre-let-7 was observed. These findings suggest that alg-1/alg-2 activities may be more important for the stability or function of let-7 stRNA than for its processing from the larger form. Alternatively, alg-1/alg-2 might also be involved in let-7 processing but the let-7 long form may be less stable, so that unprocessed let-7 does not accumulate in the absence of alg-1/alg-2 activity (Grishok, 2001).
Thus, the efficient processing of the lin-4 and let-7 stRNAs from larger precursors depends on the activity of DCR-1, a C. elegans homolog of the Drosophila multifunctional RNase III related protein, Dicer, that has been shown in Drosophila cell extracts to process dsRNA into siRNAs that can mediate RNAi (Bernstein, 2001). Further, alg-1 and alg-2, two homologs of the RNAi pathway gene rde-1, are required for efficient stRNA expression, and along with dcr-1 function to promote lin-4 and let-7 activities in temporal development. Thus, the expression of the tiny RNAs that mediate RNAi and developmental gene regulation appear to share a requirement for DCR-1 activity, while RDE-1 and its homologs provide parallel functions in these pathways. These findings are consistent with a model in which members of the RDE-1 and DCR-1 families act not only in gene silencing but also with naturally expressed dsRNAs to execute cellular and developmental gene regulatory events (Grishok, 2001).
Although there are compelling similarities between RNAi and developmental regulation by lin-4 and let-7 there are also several important differences. In RNAi, the dsRNAs utilized, typically contain long stretches of perfect base pairing. The stRNA precursors, however, are predicted to contain at most 6, for lin-4, and 13, for let-7, uninterrupted Watson-Crick base pairs. Whereas cleavage of the perfectly base-paired RNAs that initiate RNAi yields both sense and antisense, or potentially double-stranded siRNAs, only one strand of the lin-4 and let-7 stRNAs is detected. Thus, after generation of the mature stRNA, the remaining sequences must undergo rapid degradation (Grishok, 2001).
The RNAi and stRNA pathways also appear to induce distinct outcomes: RNA destruction versus translation inhibition. In RNAi the target mRNA is rapidly degraded. Although the RNase responsible for target RNA destruction is not yet known, it is thought that the antisense strand of the siRNA acts as a guide in mRNA destruction, by base-pairing with the target mRNA. The stRNAs also specifically downregulate the expression of their target genes. Although details of the mechanism by which stRNAs cause decreased expression are unknown, the regulation of lin-14 by lin-4 occurs at the translational level. Upon expression of lin-4 RNA, the levels of LIN-14 protein rapidly decline, but lin-14 mRNA levels remain constant and appear to remain associated with polyribosomes. Because let-7-mediated regulation of LIN-41 protein expression may only occur in a subset of cells, it is, as yet, unclear if the mRNA levels or polyribosome loading of this target is affected by the expression of let-7 RNA (Grishok, 2001).
The distinction between mRNA destruction by RNAi and inhibition of translation by the lin-4 regulatory RNA could reside in the target mRNA sequence or in the particular region of the mRNA targeted. Whereas siRNAs can target sequences anywhere in the mature mRNA, stRNAs pair with specific sites in the 3'UTRs of their target genes. And just as the precursors of the stRNAs have imperfect internal complementarity, the stRNAs contain imperfect complementarity to their target sequences. Imperfect pairing could permit access to RNA nucleotides by sequence-specific RNA binding proteins, or conversely, might reduce the affinity with which a nuclease could cleave the mRNA/stRNA hybrid. Alternatively, both siRNAs and stRNAs may induce similar modifications of their target mRNAs while flanking sequences provide for context dependent interactions that cause inhibition of translation in the case of lin-14 but promote destruction of other mRNAs (Grishok, 2001).
There are 24 members of the RDE-1/AGO1/PIWI family in C. elegans. The degree of conservation between certain members of this family is striking. For example, ALG-1 and ALG-2 exhibit 41% identity with AGO1 from Arabidopsis and 67%-69% identity with AGO1 relatives in animals. The common ancestor of worms and humans appears to have had both an AGO1 ortholog and a second, already-divergent family member that has given rise to the PIWI family of genes. The fact that divergent members of this family, including rde-1, qde-2, and ago-1, all function in gene silencing suggests that PTGS mechanisms represent an important ancestral function of genes within this family (Grishok, 2001).
Developmental functions have also been reported for members of the piwi and ago1 families in both animals and plants. One feature that emerges from studies of these developmental phenotypes is that many of these genes appear to regulate germ cell and stem cell functions. Perhaps germ cells and stem cells have developed PTGS mechanisms for suppressing viral and transposon pathogens that might otherwise degrade the genome and, thus, the totipotency of these cells. The developmental phenotypes associated with mutations in members of the rde-1 gene family could thus reflect a general loss of gene silencing important for stem cell maintenance or differentiation. However, the findings reported in this study suggest an alternative possibility. rde-1-related genes, alg-1 and alg-2, function with natural small RNA cofactors in specific developmental gene regulation events. Thus, it is speculated that the Drosophila genes piwi, aubergine, and ago1, the Arabidopsis gene ago1, and perhaps many other members of this family in C. elegans and other organisms may similarly have small endogenous RNA cofactors with which they function to regulate specific target mRNAs (Grishok, 2001).
While there are 24 members of the rde-1/Argonaute gene family in C. elegans, there are fewer in Arabidopsis, humans, and Drosophila. Only the Piwi and Argonaute subtypes are conserved in many species, while RDE-1 as well as most of the other C. elegans family members are more divergent. Perhaps the family of tiny RNAs that may act with these proteins has also undergone expansion in C. elegans. Whether the ancestral function of RDE-1-related genes was in developmental control or sequence-directed immunity, it is clear that a great potential exists for exploiting these proteins, along with small RNAs as guides, to direct the regulation of specific gene targets in the cell (Grishok, 2001).
Previous work has indicated that RDE-1 plays an upstream role in the initiation of interference in response to dsRNA in C. elegans. Findings described in this study suggest that ALG-1 and ALG-2 may play a similar upstream role in the lin-4 and let-7 stRNA pathways. Thus, distinct members of the extended family of RDE-1 homologs in C. elegans may play specific roles in RNAi and stRNA pathways. It is speculated that one or more of the other C. elegans RDE-1 family members may provide a similar function in cosuppression in C. elegans. One attractive possibility is that these diversified factors provide specificity to their respective pathways. This might involve a role in the recognition of the distinct trigger sequences or in insuring that the processed small RNAs are assembled into distinct downstream complexes. Perhaps members of the RDE-1 family remain associated with the RNA sequences throughout processing and provide specificity needed to ensure that the small RNAs produced are targeted to the appropriate downstream complex, for example, to mediate mRNA destruction versus translation inhibition (Grishok, 2001).
A role for RDE-1 family members in both small RNA production and targeting could explain why the inhibition of alg-1/alg-2 induces such a dramatic effect on lin-4 and let-7 function while at best reducing but not eliminating the processed stRNA. Similarly, recent studies of small RNA accumulation during RNAi suggest that rde-1 is not essential for small RNA production after exposure to dsRNA and yet rde-1(+) activity is absolutely required for interference. Conceivably, dsRNA processing might still occur in the absence of RDE-1 or its homologs but the resulting siRNAs or stRNAs may not be assembled into the appropriate downstream complexes and therefore fail to function. Nevertheless, the finding that alg-1/alg-2(RNAi) dramatically affects the accumulation of the lin-4 precursor supports a role for these factors either upstream of, or at the same step as DCR-1 (Grishok, 2001).
This study shows that dcr-1 is an essential gene and is also required for RNAi in C. elegans. dcr-1, which appears to be a single copy gene in C. elegans, could play a role in dsRNA processing important in many gene silencing and developmental pathways. DCR-1 has several motifs that might be expected in a dsRNA processing enzyme, including a helicase, a dsRNA binding domain, and two RNase III type dsRNA exonuclease domains. Thus, it is proposed that DCR-1 functions in multiple pathways important for developmental and PTGS mechanisms, and may be guided in its processing of distinct substrates by members of the RDE-1 family. Consistent with a relatively specific role for dcr-1, it was found that mature ribosomal RNAs, which are also produced by RNase III type processing, accumulate to normal levels in animals with reduced dcr-1 activity (Grishok, 2001).
The combination of a maternally provided dcr-1 activity and zygotic sterility make it difficult to unambiguously answer the question of whether this protein is absolutely essential for RNAi and stRNA pathways. Nevertheless, the reiteration of L2 fates revealed by the seam cell lineage analysis of dcr-1(RNAi) animals, and the suppression of those phenotypes by mutations in lin-14 or lin-41 are unique phenotypic and genetic signatures that strongly support the model where lin-4 and let-7 processing is dependent on dcr-1(+) activity. Perhaps the embryonic and larval lethal phenotypes associated with dcr-1 inhibition and the developmental phenotypes associated with the Arabidopsis homolog, caf 1, reflect a role for members of this gene family in the processing of other as yet unidentified small regulatory RNAs. Thus, tiny RNAs may function in a broader range of gene regulatory and developmental events than the temporal transitions mediated by the founding members of the class, the lin-4 and let-7 stRNAs (Grishok, 2001).
Double-stranded RNAs can suppress expression of homologous genes through an evolutionarily conserved process named RNA interference (RNAi) or post-transcriptional gene silencing (PTGS). One mechanism underlying silencing is degradation of target mRNAs by an RNP complex, which contains ~22 nt of siRNAs as guides to substrate selection. A bidentate nuclease called Dicer has been implicated as the protein responsible for siRNA production. This study characterizes the C. elegans ortholog of Dicer (K12H4.8; dcr-1) in vivo and in vitro. dcr-1 mutants show a defect in RNAi. Furthermore, a combination of phenotypic abnormalities and RNA analysis suggests a role for dcr-1 in a regulatory pathway comprised of small temporal RNA (let-7) and its target (e.g., lin-41) (Ketting, 2001).
A signature feature of RNA interference and related gene silencing phenomena is the production of small, ~22-nt RNAs termed guide RNAs or siRNAs. These have been observed in plants undergoing cosuppression or virus-induced gene silencing and in C. elegans and Drosophila during RNA interference. In Drosophila, biochemical studies have indicated that siRNAs are produced by nucleolytic digestion of the dsRNA silencing trigger. To test whether siRNAs are produced by an evolutionarily conserved mechanism, a combination of biochemical and genetic approaches was undertaken (Ketting, 2001).
Extracts were prepared from C. elegans embryos and these were tested for Dicer activity, as evidenced by the ability to process long dsRNA into siRNAs. Such an activity is clearly detectable. A comparison of reactions performed in parallel shows that Drosophila and C. elegans extracts produce siRNA species of different sizes. In C. elegans, siRNAs produced in vitro are 23bp in length, consistent with the size of siRNAs found in vivo. Drosophila siRNAs produced in vitro are predominantly 21 bp in length and comigrate with siRNAs that are associated with the RISC enzyme in S2 cells (Hammond, 2000). As was observed with Drosophila Dicer, longer dsRNAs are processed more efficiently. This correlates with the observation that long dsRNAs are more effective inducers of RNAi than are short dsRNAs (Ketting, 2001 and references therein).
The genome of C. elegans encodes a possible ortholog of the Drosophila Dicer protein, K12H4.8, which shares with Dicer a predicted domain structure comprising (from the N terminus to the C terminus) a helicase domain, a PAZ domain (Cerutti, 2000), dual RNAse III domains, and a double-stranded RNA-binding domain. A polyclonal antiserum was raised to the C terminus of K12H4.8. This antiserum specifically immunoprecipitates from C. elegans embryo extracts an activity that digests dsRNA into siRNAs. These results suggest that K12H4.8 is indeed the functional ortholog of Drosophila Dicer, and therefore this gene is referred to as dcr-1 (Ketting, 2001).
DCR-1, like Drosophila Dicer (Bernstein, 2001), requires ATP for efficient cleavage, and ATP hydrolysis further enhances siRNA production. It has been hypothesized that ATP hydrolysis by the helicase domain might drive a processive cleavage of dsRNA substrates by Dicer (Bernstein, 2001). A prediction of this model is that examination of reaction intermediates might reveal a ladder of products. Indeed, 500-bp dsRNA is shortened by DCR-1 in increments of ~23 nt. Reactions performed in extracts depleted of ATP produce only the first decrement of the ladder. Furthermore, when a partial dsRNA is offered as substrate, the reaction terminates at the point where the RNA becomes single-stranded. These results therefore suggest that DCR-1 converts dsRNA into siRNAs through a processive processing reaction, extracting energy for translocation along the dsRNA from ATP hydrolysis. This proposed mechanism is consistent with the observation that in vitro RNAi in Drosophila embryo extracts leads to cleavage of mRNA at characteristic ~22-nt intervals (Ketting, 2001).
To test the involvement of C. elegans Dicer in RNAi in vivo, a deletion mutant of dcr-1 was isolated. Thus far, screens for RNAi-resistant mutants have yielded viable and fertile mutants. Animals homozygous for the dcr-1 deletion are, however, sterile. Oocytes are abnormal, and no fertilized eggs were detected. These defects can be partially rescued by introduction of a transgene containing a wild-type copy of dcr-1. Fertilized eggs are formed in the presence of this transgene, but the eggs do not hatch, presumably because of loss of the transgene or limited expression of DCR-1 from the transgene in the germ line (Ketting, 2001).
To test whether dcr-1 animals are resistant to RNAi, these worms were fed dsRNA homologous to the unc-22 gene. Surprisingly, a wild-type RNAi response was observed. This might indicate that RNAi can proceed independently of Dicer activity. Alternatively, maternally contributed DCR-1 protein might be sufficient to generate an RNAi response. In fact, at least one other gene required for RNAi (mut-15) displays such a maternal effect. It was expected that maternally contributed Dicer protein would be absent from the germ line of adult dcr-1 homozygotes. It was therefore tested whether RNAi could be used to silence a GFP transgene that is expressed specifically in that tissue. Although RNAi is quite effective at silencing germ-line GFP in wild-type worms, dcr-1 homozygotes are RNAi-resistant, and it is concluded that Dicer is essential for RNAi in at least some tissues. The possibility that there is a second pathway that induces RNAi in the absence of Dicer cannot be excluded; however, the most parsimonious explanation is preferred: that the residual RNAi effects result from persistence of maternal Dicer protein (Ketting, 2001).
In addition to being sterile, dcr-1 homozygotes show a number of additional phenotypic abnormalities. Among these is a defect of the seam cells in the L4-to-adult transition. These cells fail to fuse, and in some cases undergo an additional round of cell division. As a result, the alae are absent in ~60% (38/62) of the dcr-1(pk1531) homozygous animals. Interestingly, this phenotype is also characteristic of loss-of-function mutations in the let-7 gene (Ketting, 2001).
The let-7 gene product (Drosophila homolog: let-7) is a small, noncoding RNA that regulates the timing of developmental events in C. elegans (therefore named small temporal RNA or stRNA. Of interest, the let-7 RNA is 21 nt in length, and it has been hypothesized that the let-7 RNA is produced by post-transcriptional processing of a longer precursor that is predicted to form an extended hairpin structure, which may be a substrate for DCR-1. Regulation by let-7 occurs at the translational level and presumably is mediated by complementary base-pairing between let-7 and the 3'-untranslated regions of target genes (Ketting, 2001 and references therein).
One of the in vivo targets of let-7 is lin-41 (Drosophila homolog: dappled), and the increased expression of this protein in let-7 mutants leads to the burst vulva phenotype. Interestingly, dcr-1 homozygous mutants also display a burst vulva phenotype, up to 80% (17/21), which can be rescued by introducing the wild-type dcr-1 gene. Tests were performed to see if this phenotype can be partially suppressed by down-regulating LIN-41 protein through RNAi, and indeed it can -- after RNAi of lin-41 only 25% burst vulva (5/20) are found. This suggests that the burst vulva phenotype in dcr-1 mutant animals is at least partially caused by an up-regulation of LIN-41, and the epistatic effect is an indication that dcr-1 and lin-41 indeed act in the same pathway. Conversely, hypomorphic alleles of lin-41 have an Egl phenotype (an egg-laying defect), whereas null alleles of lin-41 are sterile owing to the absence of oocytes. Accordingly, different levels of ectopic expression of DCR-1 might, via down-regulation of lin-41, induce an Egl phenotype or sterility. This is indeed what is found. Although the phenotypes described above are not specific enough to directly imply dcr-1 as an actor in the let-7/lin-41 pathway, the phenotypic relationship between animals with altered DCR-1 levels and animals with alterations in the let-7/lin-41 pathway, are suggestive (Ketting, 2001).
To test this more directly two approaches were undertaken (1) Using Drosophila embryo extracts and immunoprecipitates as a source of Dicer, tests were performed to see whether Dicer could process Drosophila let-7 precursor RNA into its mature form in vitro. Indeed, the ~75-nt hairpin was processed into an ~21-nt mature RNA with a disproportionately high efficiency as compared to perfect duplexes of similar size. (2) It was asked whether the dcr-1 mutation had an effect on the levels of let-7 RNA in vivo. Levels of mature let-7 RNA are reduced in dcr-1 mutant animals, and that this reduction is accompanied by an accumulation of the longer let-7 RNA precursor. Together these approaches show that dcr-1 is directly involved in the conversion of the double-stranded let-7 precursor RNA into the active, 21-nt species (Ketting, 2001).
RNAi and PTGS can clearly function to protect the genome against viruses and transposons. In addition, there is some evidence that factors involved in RNAi or PTGS also play a role in proper germ-line development. This study has shown that at least one component of the RNAi machinery in animals, Dicer, also plays a role in generating small RNAs involved in developmental timing (Ketting, 2001).
The mechanisms by which RNAi and stRNAs regulate the expression of target genes are quite distinct. In the former case, mRNAs are destroyed, whereas in the latter, expression is inhibited at the translational level. This raises the possibility that 22-nt RNAs produced by Dicer might act in multiple, distinct regulatory pathways that are not otherwise mechanistically related. Alternatively, the effector machinery may be shared by both processes, with an altered outcome of target recognition. The let-7 RNA is not perfectly homologous to its target substrates, and such a mismatch may inhibit the ability of RISC to cleave its substrates, effectively switching the mode of regulation from degradation to translational repression. It should be noted that let-7 is, most likely, not the only substrate for Dicer that is required for normal development. There may be many other endogenously encoded dsRNAs that are processed by Dicer to produce stRNA molecules, for example, lin-4. For this gene it has been shown that the mismatch between lin-4 and its target is critically required for proper regulation (Ketting, 2001 and references therein).
The 22-nt siRNAs that act in RNAi/PTGS have been found in multiple species. Dicer activity has been detected in extracts of plants and fungi (Nicotiana benthamiana, Neurospora crassa N. crassa and Phytophthora infestans). Thus far, stRNA genes (like let-7) have been identified in animals ranging from C. elegans to humans. Considered together, these observations suggest conserved roles for Dicer proteins in both dsRNA-induced silencing and in regulating developmental timing. Ironically, small temporal RNAs have yet to be identified in plants, in which developmental defects have been associated with mutants in the RNAi machinery. It will therefore be of interest to determine whether small temporal RNAs also regulate developmental timing in plants and to investigate whether this mode of gene regulation might also extend to nondevelopmental programs of gene expression (Ketting, 2001).
Double-stranded (ds) RNA induces potent gene silencing, termed RNA interference (RNAi). At an early step in RNAi, an RNaseIII-related enzyme, Dicer (DCR-1), processes long-trigger dsRNA into small interfering RNAs (siRNAs). DCR-1 is also required for processing endogenous regulatory RNAs called miRNAs, but how DCR-1 recognizes its endogenous and foreign substrates is not yet understood. The C. elegans RNAi pathway gene, rde-4, encodes a dsRNA binding protein that interacts during RNAi with RNA identical to the trigger dsRNA. RDE-4 protein also interacts in vivo with DCR-1, RDE-1, and a conserved DExH-box helicase. These findings suggest a model in which RDE-4 and RDE-1 function together to detect and retain foreign dsRNA and to present this dsRNA to DCR-1 for processing (Tabara, 2002).
RNA interference (RNAi) is the process of long, double-stranded (ds), RNA-dependent posttranscriptional gene silencing (PTGS). In lower eukaryotes, dsRNA introduced into the cytoplasm is cleaved by the RNaseIII-like enzyme, Dicer, to 21-23 nt RNA (short interfering [si] RNA), which may serve as guide for target mRNA degradation. In mammals, long-dsRNA-dependent PTGS is applicable only to a limited number of cell types, whereas siRNA synthesized in vitro is capable of effectively inducing gene silencing in a wide variety of cells. Although biochemical and genetic analyses in lower eukaryotes show that Dicer and some PIWI family member proteins are essential for long-dsRNA-dependent PTGS, little is known about the molecular mechanisms underlying siRNA-based PTGS. Dicer and eIF2C translation initiation factors belonging to the PIWI family (eIF2C1-4) play an essential role in mammalian siRNA-mediated PTGS, most probably through synergistic interactions. Immunoprecipitation experiments suggest that, in human and mouse cells, complex formation occurs between Dicer and eIF2C1 or 2 and that the PIWI domain of eIF2C is essential for the formation of this complex (Doi, 2003).
RNA interference is implemented through the action of the RNA-induced silencing complex (RISC). Although Argonaute2 has been identified as the catalytic center of RISC, the RISC polypeptide composition and assembly using short interfering RNA (siRNA) duplexes has remained elusive. RISC is shown to be composed of Dicer, the double-stranded RNA binding protein TRBP, and Argonaute2. This complex can cleave target RNA using precursor microRNA (pre-miRNA) hairpin as the source of siRNA. Although RISC can also utilize duplex siRNA, it displays a nearly 10-fold greater activity using the pre-miRNA Dicer substrate. RISC distinguishes the guide strand of the siRNA from the passenger strand and specifically incorporates the guide strand. Importantly, ATP is not required for miRNA processing, RISC assembly, or multiple rounds of target-RNA cleavage. These results define the composition of RISC and demonstrate that miRNA processing and target-RNA cleavage are coupled (Gregory 2005).
This study shows that, although RISC could utilize the 22 nt duplex as the source of the siRNA, it displays far greater activity once a pre-miRNA, a substrate of Dicer, is used as the source of siRNA. These results strongly support the contention that Dicer cleavage activity is tightly coupled into the effector step of RNAi mediated by Ago2. The coupling of the two enzymatic activities makes ample biological sense since, once the duplex RNA is cleaved by Dicer, it could be unwound and handed over to Ago2 for target-RNA cleavage in a concerted reaction. The data showing a physical and functional coupling of pre-miRNA processing and RISC assembly also provide a mechanistic framework that explains the observations that 27 nt double-stranded RNAs or short hairpin RNAs, both of which are Dicer substrates, are considerably more potent triggers of RNAi than the short duplex siRNA. This study shows that, although RISC can utilize the 22 nt duplex as the source of the siRNA, it displays far greater activity once a pre-miRNA, a substrate of Dicer, is used as the source of siRNA. These results strongly support the contention that Dicer cleavage activity is tightly coupled into the effector step of RNAi mediated by Ago2. The coupling of the two enzymatic activities makes ample biological sense since, once the duplex RNA is cleaved by Dicer, it could be unwound and handed over to Ago2 for target-RNA cleavage in a concerted reaction. The data showing a physical and functional coupling of pre-miRNA processing and RISC assembly also provide a mechanistic framework that explains the observations that 27 nt double-stranded RNAs or short hairpin RNAs, both of which are Dicer substrates, are considerably more potent triggers of RNAi than the short duplex siRNA (Gregory, 2005)
MicroRNAs (miRNAs) are small RNAs that regulate gene expression posttranscriptionally. To block all miRNA formation in zebrafish, maternal-zygotic dicer (MZdicer) mutants were generated 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 (Giraldez, 2005).
MicroRNAs are evolutionarily conserved small non-protein-coding RNA gene products that regulate gene expression at the posttranscriptional level. In animals, mature miRNAs are ~22 nucleotides (nt) long and are generated from a primary transcript (termed pri-miRNA) through sequential processing by nucleases belonging to the ribonuclease III (RNaseIII) family. Initially, Drosha cleaves the pre-miRNA and excises a stem-loop precursor of ~70 nt (termed pre-miRNA), which is then cleaved by Dicer. One strand of the processed duplex is incorporated into a silencing complex and guides it to target sequences. This results in the cleavage of target mRNAs and/or the inhibition of their productive translation (Giraldez, 2005).
Several hundred vertebrate miRNAs and several thousand miRNA targets have been predicted or identified, but little is known about miRNA function during development. Clues to vertebrate miRNA function have come from several approaches, including expression analyses, computational prediction of miRNA targets, experimental support of predicted targets, and gain-of-function approaches. These studies have led to the suggestions that vertebrate miRNAs might be involved in processes such as stem cell maintenance or cell fate determination; however, no loss-of-function analysis has assigned a role for a particular miRNA or miRNA family in vivo, and it has been unclear how widespread the role of miRNAs is during vertebrate embryogenesis (Giraldez, 2005).
One approach to reveal the global role of vertebrate miRNAs is to abolish the generation of mature miRNAs with the use of dicer mutants. For example, dicer mutant embryonic stem cells fail to differentiate in vivo and in vitro, and dicer mutant mice die before axis formation, suggesting that mature miRNAs (or other Dicer products) are essential for early mammalian development. In zebrafish, maternal dicer activity has hampered the analysis of the single dicer gene. Mutants for the zygotic function of dicer (Zdicer) retain pre-miRNA processing activity up to 10 days postfertilization, presumably because of maternally contributed dicer. Zdicer mutants have no obvious defects other than a developmental delay at 7 to 10 days postfertilization, a stage when embryogenesis and major steps of organogenesis have been achieved. Hence, the global role of miRNAs during vertebrate embryogenesis is unknown. In light of these observations, zebrafish embryos were generated that lack both maternal and zygotic dicer activity (Giraldez, 2005).
Similar to other model systems, wild-type zebrafish embryos generate mature miRNAs from endogenous or exogenously provided pri-miRNAs, resulting in the post-transcriptional repression of reporter genes. miRNAs induce the cleavage of reporter RNAs with perfectly complementary target sites (PT) in the 3' untranslated region (3'UTR), whereas imperfectly complementary sites (IPT) result in the noneffective translation of reporter mRNAs. Previous biochemical and genetic studies have shown that Dicer is required for the generation of mature miRNAs. To determine whether MZdicer embryos lack mature miRNAs, total RNA from 1-day-old zebrafish embryos was hybridized to a microarray of probes for 120 different zebrafish mature miRNAs. Although such arrays are susceptible to cross-hybridization artifacts, a marked reduction was observed of signals in MZdicer mutants compared with wild-type embryos and zygotic dicer mutants. Of the 120 miRNA probes, 59, 35, and 9 gave a detectable signal in wild-type embryos, Zdicer mutants, and MZdicer mutants, respectively. To test for the presence of mature miRNAs more specifically, Northern blot analyses were performed. Of eight miRNAs present in wild-type embryos, none was detected in MZdicer mutants. These and other experiments suggested that mature miRNAs were not generated in MZdicer mutants (Giraldez, 2005).
The absence of mature miRNAs in MZdicer mutants allowed a determination of their global requirement during early zebrafish development. The MZdicer phenotype notably differs from that of Zdicer mutants, which are indistinguishable from wild-type embryos during these stages. Morphological analysis during the first 5 days of development revealed that axis formation and the regionalization of MZdicer mutants were intact. Major subregions and cell types were present, ranging from forebrain, eye, midbrain, hindbrain, ear, pigment cells, and spinal cord to hatching gland, heart, notochord, somites, and blood. In contrast, morphogenetic processes during gastrulation, somitogenesis, and heart and brain development were severely affected. MZdicer mutants also developed more slowly than wild-type embryos, with 3 to 4 hours of delay within the first 24 hours of development (Giraldez, 2005).
During zebrafish gastrulation, four concomitant cell rearrangements take place: (1) epiboly (spreading of the embryo over the yolk, (2) internalization (formation of mesodermal and endodermal germ layers, (3) convergence (movement of cells toward the dorsal side), and (4) extension (lengthening of the embryo). MZdicer mutants fail to coordinate epiboly and internalization. This results in mutant embryos that had undergone prechordal plate migration corresponding to 80% epiboly in wild-type embryos, yet epiboly movements are delayed to a stage equivalent to 50% to 60% epiboly. MZdicer embryos also display a reduced extension of the axis, resulting in a shortening of the embryo and an accumulation of cells in the head region. Later during development, MZdicer mutants have a reduced posterior yolk extension (Giraldez, 2005).
Neurulation is severely affected in MZdicer embryos. The mutant neural plate gives rise to the neural rod, but the subsequent formation of the neurocoel and neural tube is notably impaired. The formation of the brain ventricles is severely reduced. In wild-type embryos, several constrictions subdivide the brain into distinct regions. These constrictions do not form in MZdicer mutants. For example, the midbrain-hindbrain boundary that is very prominent in wild-type embryos does not form in MZdicer mutants. In addition, retinal development is affected. Defects in the spinal cord are manifested by a rudimentary neurocoel and a reduction of the floor plate in the trunk (Giraldez, 2005).
Despite the gross morphological malformations of the nervous system, gene expression analysis suggested that anterior-posterior and dorsal-ventral patterning are not severely disrupted. Analysis of anterior-posterior and dorsal-ventral markers revealed normal specification of the optic stalk, forebrain, midbrain-hindbrain boundary, otic vesicles, hindbrain rhombomeres, and the dorsal and ventral neural tube (Giraldez, 2005).
Analysis of neuronal differentiation and axonal markers, with the use of HuC and HNK antibodies, revealed mispositioned trigeminal sensory neurons adjacent to the eye. In addition, defasciculation of the postoptic commissure was observed in MZdicer embryos. In the hindbrain, multiple neurons project longitudinal axons anteriorly and posteriorly and form a ladder-like structure on each side of the midline. This scaffold is disrupted and defasciculated in MZdicer mutants, but longitudinal axonal projections are established. In addition, touch-induced escape behavior is severely diminished in MZdicer mutants. Taken together, these results indicate that early patterning and fate specification in the embryonic nervous system are largely unaffected by lack of miRNAs. In contrast, normal brain morphogenesis and neural differentiation and function require Dicer activity (Giraldez, 2005).
During somitogenesis, the paraxial mesoderm becomes segmented. MZdicer embryos formed normally spaced somites and express the muscle marker myoD similar to wild-type embryos. Later in development, the somites acquire a chevron shape in wild-type embryos but form irregular boundaries in MZdicer mutants. Endothelial and hematopoietic precursor cells are present as judged from the expression of the markers fli-1 and scl, respectively, but endocardial fli-1 expression is reduced and blood circulation disrupted in MZdicer mutants. Analysis of the markers pax2a, GFP-nanos-3'UTR, fkd1, cmlc2, and fkd2 revealed that pronephros, germ cells, endoderm, cardiomyocytes, and liver cells, respectively, are specified. MZdicer mutants have contractile cardiomyocytes but the two chambers characteristic of the wild-type heart do not form; instead, a tubular heart and pericardial edema developed (Giraldez, 2005).
Taken together, these results indicated that MZdicer mutant embryos are patterned correctly and have multiple specified cell types but underwent abnormal morphogenesis, in particular during neural development and organogenesis (Giraldez, 2005).
To identify miRNAs that might play important roles during early zebrafish development, small RNAs (~18 to 28 nt) were cloned from eight developmental stages between fertilization and 48 hours of development. These experiments identified miR-430a, miR-430b, and miR-430c as three highly expressed miRNAs, as well as several related species, miR-430d to miR-430h, which were expressed at lower levels. The miR-430 family members each had the same sequence at a segment encompassing nucleotides 2 to 8; this segment is known as the 'seed' and has been shown to be the miRNA segment most important for target recognition. The family members also have strong homology in their 3' region, but differ in their central and terminal nucleotides. Mapping of the miR-430 family to the zebrafish genome revealed a locus composed of multiple copies of the miR-430a,c,b triplet, with more than 90 copies of the miRNAs within 120 kb. miRNA genes are sometimes observed in clusters of about two to seven, which are frequently transcribed as a single polycistronic transcript, but the zebrafish miR-430 cluster has many more miRNAs than reported in other clusters. The miR-430 miRNAs are conserved and clustered in other fish genomes, including Fugu rubripes and Tetraodon nigroviridis. The miR-430 miRNAs belong to a superfamily that includes the vertebrate miR-17-miR-20 family, found in much smaller clusters in mammalian genomes. Despite the sequence similarities of the two families, members of the miR-17-miR-20 family derive from the opposite arm of their precursors; this suggests convergent rather than divergent origins of the two families. The miR-430 RNAs might share evolutionary origins with some of the miRNAs expressed specifically in mammalian embryonic stem cells, including miR-302 and miR-372, which have the same seed nucleotides and derive from the same arm of the hairpin (Giraldez, 2005).
The miR-430 miRNAs are initially expressed at about 50% epiboly [5 hours postfertilization (hpf)], continue to be expressed during gastrulation and somitogenesis, and then decline at about 48 hpf. Analysis of GFP sensors with perfect target sites for miR-430a or miR-430b suggested that the miR-430 miRNAs are ubiquitously expressed and active during early development (Giraldez, 2005).
miRNA duplexes are still active in MZdicer mutants. This allowed a determination of whether aspects of the MZdicer mutant phenotype can be suppressed by providing specific miRNAs that are normally expressed during early zebrafish development (miR-1, miR-204, miR-96, miR-203, miR-430a, miR-430b, or miR-430c). It was also reasoned that such rescue would unequivocally demonstrate that a particular phenotype is caused by the loss of a specific mature miRNA and not by the lack of small interfering RNAs (siRNAs) or the abnormal accumulation of pre-miRNAs in MZdicer mutants. It was found that injection of miR-430 duplexes (miR-430a, miR-430b, or miR-430c) rescues the brain morphogenesis defects in MZdicer mutants. This rescue is specific, as indicated by two control experiments. (1) Injection of unrelated miRNA duplexes did not cause any rescue. (2) Injection of a miRNA duplex with two point substitutions in the 5' seed did not rescue the MZdicer phenotype. Rescue of MZdicer mutant embryos by miR-430 (MZdicer+miR-430) results in normal brain ventricles and brain constrictions. For example, the midbrain-hindbrain boundary forms in MZdicer+miR-430 as in wild-type embryos. Injection of miR-430 also induces a substantial rescue of the neuronal defects observed in MZdicer mutants. MZdicer+miR-430 also display partially rescued gastrulation, retinal development, somite formation, and touch response. In contrast, the defects in the development of the ear and heart and the lack of circulation were not rescued. Later during development (90 hpf), MZdicer+miR-430 embryos are developmentally delayed and display reduced growth similar to MZdicer. These results indicate that loss of miR-430 miRNAs accounts for some but not all of the defects observed in MZdicer embryos (Giraldez, 2005).
This study of zebrafish that lack Dicer RNaseIII activity and mature miRNAs provides three major insights into the roles of miRNAs during embryogenesis. (1) The results suggest that mature miRNAs do not have widespread essential roles in fate specification or signaling during early zebrafish development. Phenotypic comparison between MZdicer mutants and embryos with aberrant signaling pathways (Nodal, Hedgehog, Wnt, Notch, CXCR4, FGF, BMP, retinoic acid, or STAT3) suggests that none of these pathways is markedly affected by the absence of miRNAs. For example, MZdicer mutants do not display the phenotypes seen upon an increase or decrease in Nodal or BMP signaling. This suggests that miRNAs might have modulating or tissue-specific rather than obligatory roles in various signaling pathways. Similarly, this study reveals that MZdicer mutants can differentiate multiple cell types during development. This suggests that mature miRNAs are not required to specify the major embryonic cell lineages in zebrafish. The results do not exclude more specific roles in fate specification, such as modulating the choice between highly related cell fates. For example, lsy-6 in Caenorhabditis elegans controls the distinction between two closely related neurons, and mouse miR-181 seems to regulate the ratio of cell types within the lymphocyte lineage. miRNAs might also function at later stages to stabilize and maintain a particular fate. For instance, miRNAs might repress large numbers of target mRNAs to maintain tissue homeostasis by dampening fluctuations in gene expression. However, the transplantation results argue against an absolute requirement for miRNAs in every cell type. In particular, fertile adults were generated from MZdicer mutant donors by germ cell transplantation. This indicates that primordial germ cells, the ultimate stem cells, proliferate and remain pluripotent to form the adult germ line in the absence of miRNAs. Multigeneration transplantation studies are required to determine if the lack of miRNAs has effects on germ cell maintenance. More exhaustive analysis of different cell types and signaling pathways is needed to test for more subtle or later roles of miRNAs in zebrafish, but the current study excludes a general role in signaling, embryonic fate specification, or germ line stem cell development (Giraldez, 2005).
(2) The results suggest important roles for miRNAs during embryonic morphogenesis and differentiation, ranging from epiboly and somitogenesis to heart, ear, and neural development. For example, loss of Dicer leads to defects in the positioning of neurons, the defasciculation of axons, and impaired touch-induced behaviors. Most notably, mutants form a neural rod but fail to generate normal brain ventricles. In addition, the morphological constrictions that subdivide the anterior-posterior axis do not form in the absence of Dicer, despite the regionalization observed by marker analysis. These results reveal essential roles of miRNAs during zebrafish morphogenesis (Giraldez, 2005).
(3) This study identified a previously unknown miRNA family, the absence of which is likely to account for the brain morphogenesis defects in MZdicer mutants. The miR-430 family has more genes than any miRNA family described to date, is conserved in fish, and is part of a superfamily found in other vertebrates. Injection of miR-430 duplexes suppresses the brain morphogenesis defects in MZdicer mutants. This complementation approach can now be applied to determine which miRNAs (or siRNAs) account for the MZdicer phenotypes that cannot be rescued by miR-430. The miR-430 family might inhibit mRNAs that are provided maternally or expressed during early embryogenesis but are detrimental to later steps in morphogenesis. Cell shape changes, cell rearrangements, and fluid dynamics are thought to generate both extrinsic and intrinsic forces that contribute to neural tube and ventricle formation, but the underlying molecular mechanisms are poorly understood. The study of the miR-430 family and its targets therefore provides a genetic entry point to dissect the molecular basis of brain morphogenesis (Giraldez, 2005).
Members of the ribonuclease III superfamily of double-stranded(ds)-RNA-specific endoribonucleases participate in diverse cellular RNA maturation and degradation pathways. A recently identified eukaryotic RNase III family member, named Dicer, functions in the RNA interference (RNAi) pathway by producing 21-23 bp dsRNAs that target the selective destruction of homologous RNAs. RNAi is operative in animals, plants, and fungi, where it is proposed to inhibit viral reproduction and retroposon movement, as well as to participate in developmental pathways. RNAi functions in mammalian cells, including mouse oocytes and embryos. This article reports the cDNA sequence characterization and expression analysis of the mouse Dicer ortholog. On the basis of the cDNA sequence, the Dicer polypeptide is 1906 amino acids and has a predicted molecular mass of 215 kDa. Mouse Dicer contains a DExH/DEAH helicase motif; a PAZ domain; a tandem repeat of RNase III catalytic domain sequences, and a dsRNA-binding motif. The Dicer gene maps to a single locus on the distal portion of mouse chromosome 12. The Dicer transcript is expressed from the embryonic through adult stages of development. The Dicer transcript is also present in a wide variety of adult mouse organs. The highly conserved set of functional domains and the occurrence of a single-copy gene strongly indicate that the encoded protein is the RNase III ortholog responsible for dsRNA processing in the RNAi pathway (Nicholson, 2002).
RNAi-mediated heterochromatin assembly in fission yeast requires the RNA-induced transcriptional silencing (RITS) complex and a putative RNA-directed RNA polymerase (Rdp1). 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 (Motamedi, 2004).
RNA interference is a conserved mechanism by which double-stranded RNA is processed into short interfering RNAs (siRNAs) that can trigger both post-transcriptional and transcriptional gene silencing. In fission yeast, the RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. Furthermore, these analyses suggest that RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys9. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance (Noma, 2004).
RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. This study describes 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 (Verdel, 2004).
In fission yeast, factors involved in the RNA interference (RNAi) pathway including Argonaute, Dicer, and RNA-dependent RNA polymerase are required for heterochromatin assembly at centromeric repeats and the silent mating-type region. RNA-induced initiation of transcriptional gene silencing (RITS) complex containing the Argonaute protein and small interfering RNAs (siRNAs) localizes to heterochromatic loci and collaborates with heterochromatin assembly factors via a self-enforcing RNAi loop mechanism to couple siRNA generation with heterochromatin formation. The role were investigated of RNA-dependent RNA polymerase (Rdp1) and its polymerase activity in the assembly of heterochromatin. Rdp1, similar to RITS, localizes to all known heterochromatic loci, and its localization at centromeric repeats depends on components of RITS and Dicer as well as heterochromatin assembly factors including Clr4/Suv39h and Swi6/HP1 proteins. A point mutation within the catalytic domain of Rdp1 abolishes its RNA-dependent RNA polymerase activity and results in the loss of transcriptional silencing and heterochromatin at centromeres, together with defects in mitotic chromosome segregation and telomere clustering. Moreover, the RITS complex in the rdp1 mutant does not contain siRNAs, and is delocalized from centromeres. These results not only implicate Rdp1 as an essential component of a self-enforcing RNAi loop but also ascribe a critical role for its RNA-dependent RNA polymerase activity in siRNA production necessary for heterochromatin formation (Sugiyama, 2005).
Dicer is the enzyme that cleaves double-stranded RNA (dsRNA) into 21-25-nt-long species responsible for sequence-specific RNA-induced gene silencing at the transcriptional, post-transcriptional, or translational level. The dicer-1 gene was disrupted in mouse embryonic stem (ES) cells by conditional gene targeting, and Dicer-null ES cells were generated. These cells are viable, despite being completely defective in RNA interference (RNAi) and the generation of microRNAs (miRNAs). However, the mutant ES cells display severe defects in differentiation both in vitro and in vivo. Epigenetic silencing of centromeric repeat sequences and the expression of homologous small dsRNAs are markedly reduced. Re-expression of Dicer in the knockout cells rescues these phenotypes. These data suggest that Dicer participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing (Kanellopoulou, 2005).
Heterochromatin formation depends on the RNAi machinery in S. pombe, Tetrahymena, and Drosophila, and links between DNA methylation and RNAi have been reported in plants. However, the original premise was that the RNAi pathway is mainly involved in PTGS. When the mechanism of RNAi was initially reported in C. elegans, it was noted that promoter and intronic sequences were ineffective in dsRNA-mediated gene silencing, thus reasoning that this process occurred post-transcriptionally. While there are examples of RNA-guided chromatin modifications in various organisms, they have, to date, been considered an oddity of the relevant host organism and not a general mechanism of epigenetic control. Evidence is reported in this study of a link between the RNAi pathway and DNA methylation of an endogenous sequence in animals, raising the possibility that RNA-based transcriptional gene silencing is a general event in higher eukaryotic gene regulation (Kanellopoulou, 2005).
Specifically, the data suggest that ablation of Dicer, a central molecule in the RNAi pathway, leads to derepression of normally silenced genetic elements, such as transposons and centromeric heterochromatin. An RNA component seems to be involved in heterochromatin formation in mammalian cells, but the specific nature of this RNA component and its possible link to the RNAi machinery have not been established. The present experiments directly address this issue. Centromeric repeat sequence-derived transcripts are up-regulated in DCR minus cells, suggesting that these regions escape transcriptional gene silencing in the absence of Dicer. However, Northern blot analyses have shown that Dicer extensively processes the largely double-stranded RNA derived from centromeric repeats into smaller RNAs, ranging from 25 to 150 nt. While the apparent lack of centromeric region silencing might reflect failure of DCR minus cells to efficiently convert primary transcripts into smaller dsRNA species, the loss of heterochromatin-related modifications in the centromeric regions of the mutants suggests another possibility. Conceivably, the 25-30-nt species, which are absent in the Dicer-deficient cells, could be analogous to small, centromeric heterochromatin-encoded RNAs cloned from S. pombe. If so, one might hypothesize that these RNAs are incorporated into a mammalian RITS complex and function as guides for TGS of homologous genomic sequences (Kanellopoulou, 2005).
Dicer is an essential component of RNA interference (RNAi) pathways, which have broad functions in gene regulation and genome organization. Probing the consequences of tissue-restricted Dicer loss in mice indicates a critical role for Dicer during meiosis in the female germline. Mouse oocytes lacking Dicer arrest in meiosis I with multiple disorganized spindles and severe chromosome congression defects. Oogenesis and early development are times of significant post-transcriptional regulation, with controlled mRNA storage, translation, and degradation. These results suggest that Dicer is essential for turnover of a substantial subset of maternal transcripts that are normally lost during oocyte maturation. Furthermore, evidence was found that transposon-derived sequence elements may contribute to the metabolism of maternal transcripts through a Dicer-dependent pathway. These studies identify Dicer as central to a regulatory network that controls oocyte gene expression programs and that promotes genomic integrity in a cell type notoriously susceptible to aneuploidy (Murchison, 2007).
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