loquacious
DEVELOPMENTAL BIOLOGY

loqs RA isoform is the predominant mRNA species in dissected testes, whereas loqs RB is the most abundant species in ovaries. Both isoforms are expressed in the carcasses of males and females after removal of the gonads. Using two independent antibodies raised against an N-terminal Loqs peptide, a candidate protein for Loqs isoform PC was detected in S2 cells (Forstemann, 2005).

Effects of Mutation or RNAi

Dicer-1 has been shown to be the pre-miRNA processing factor in Drosophila. Depletion of Dicer-1 by RNAi results in a marked accumulation of pre-miR-bantam (pre-miR-ban) (Okamura, 2004). Depletion of Loqs by RNAi results in a similar effect to Dicer-1 depletion for miR-ban. Loqs dsRNAs causes the suppression of Loqs mRNA. RNAi against Loqs does not appear to affect Dicer-1 protein levels, suggesting that the observed pre-miRNA accumulation in Loqs-depleted cells is not simply due to destabilizing Dicer-1. Similar effects on miR-8 were seen in Dicer-1- and Loqs-depleted S2 cells. Depletion of Dicer-2 and R2D2, which form the enzyme complex predominantly responsible for generating siRNAs from long dsRNA has no significant effect on pre-miRNA processing. These results show that along with Dicer-1, Loqs is essential for efficient pre-miRNA processing in vivo (Saito, 2005).

Normal microRNA maturation and germ-line stem cell maintenance requires Loquacious, a double-stranded RNA-binding domain protein

A mutant allele of CG6866, loqsf00791, was recovered in a large-scale piggyBac transposon mutagenesis screen of Drosophila. The f00791 piggyBac inserted 57 nucleotides upstream of the loqs transcription start site: although annotated as lethal, homozygous mutant loqsf00791 flies are viable but completely female sterile. Precise excision of the f00791 piggyBac transposon fully reverted the female sterility. Analysis by quantitative RT-PCR using primers that amplify all three loqs mRNA splice variants showed that somatic female loqsf00791 tissues express approximately 5-fold less loqs mRNA than wild-type, while loqsf00791 mutant ovaries express approximately 40-fold less loqs mRNA than wild-type ovaries. Testes express approximately 3-fold less loqs mRNA in the loqsf00791 mutant than in wild type. These data suggest that the mutant phenotype should be strongest in ovaries, consistent with the mutation causing female sterility as its most obvious defect (Forstemann, 2005).

To assess the function of loqs in miRNA biogenesis, total RNA was isolated from loqsf00791 males, and the steady-state levels of mature and pre-miRNA were determined for miR-277 and bantam, which are both expressed in adult tissues. A 100-fold increase in pre-miR-277 and a 12-fold increase in pre-bantam RNAs was detected in homozygous mutant loqsf00791 males, but not in heterozygous loqsf00791 or heterozygous or homozygous r2d2 mutant males. In contrast, the amount of mature miR-277 or bantam was only slightly reduced in the loqsf00791 homozygotes (Forstemann, 2005).

Since loqs mRNA expression is lowest in the ovaries of loqsf00791 mutant flies, the levels were examined of pre-miR-7 and mature miR-7, a miRNA that is expressed in whole males, manually dissected ovaries, and the female carcasses remaining after removing the ovaries. While pre-miR-7 increased in all loqsf00791 homozygous mutant tissues, relative to wild-type or loqs heterozygotes, the disruption of miR-7 production in ovaries is striking: not only does pre-miR-7 accumulate, but also mature miR-7 is dramatically reduced. These data suggest that Loqs protein function is required for the maturation of miRNA and demonstrate a direct correlation between loqs mutant allele strength and disruption of miRNA processing (Forstemann, 2005).

To confirm the function of loqs in pre-miRNA processing, cultured Drosophila S2 cells were depleted of loqs mRNA by RNAi. Eight days after incubating S2 cells with dsRNA corresponding to the first 300 nucleotides of the loqs coding sequence, the steady-state levels of pre-miRNA and mature miRNA were determined for miR-277 and bantam. Relative to an unrelated dsRNA control, dsRNA corresponding to dcr-1 caused an approximately 9-fold and approximately 23-fold increase in steady-state pre-miR-277 and bantam levels, respectively, and dsRNA corresponding to loqs caused an approximately 2-fold and approximately 6-fold increase in steady-state pre-miR-277 and bantam levels, respectively. In these experiments, RNAi of dcr-1 more completely depleted Dcr-1 protein than RNAi of loqs reduced Loqs protein. RNAi of dcr-2,r2d2, or drosha did not alter pre-miRNA levels for either miR-277 or bantam, nor did it alter Dcr-1 or Loqs levels. The Drosha/Pasha protein complex functions before pre-miRNA processing, converting primary miRNA (pri-miRNA) to pre-miRNA. Consistent with the idea that Loqs functions with Dcr-1 to convert pre-miRNA to mature miRNA, RNAi of drosha together with loqs alleviates the high pre-miRNA levels observed for RNAi of loqs alone, demonstrating that Loqs acts after Drosha (Forstemann, 2005).

Next, processing of 20 nM exogenous pre-let-7 into mature let-7 was examined in lysates from ovaries or S2 cells. Initial velocities were calculated for each reaction to permit comparison of processing rates. Lysate from homozygous loqsf00791 mutant ovaries processes pre-let-7 RNA to mature let-7 approximately 19-fold more slowly than wild-type ovary lysate. Moreover, lysate prepared from S2 cells soaked with a green fluoresecent protein (GFP) control dsRNA (GFP[RNAi]) or drosha dsRNA (drosha[RNAi]) accurately and efficiently converts exogenous pre-let-7 RNA into mature let-7. In contrast, bothdcr-1(RNAi) and loqs (RNAi) S2 cell lysates convert pre-miRNA to mature miRNA approximately 5- and approximately 4-fold, respectively, more slowly than the control lysate. Thus, Loqs is required for production in vivo of normal levels of miR-7, miR-277, and bantam, and the efficient conversion of pre-let-7 to mature let-7 in vitro. Together, these four miRNAs include both miRNAs found on the 5' and on the 3' side of the pre-miRNA stem, suggesting a general role for Loqs in pre-miRNA processing (Forstemann, 2005).

The loqsf00791 mutation causes pre-miRNAs to accumulate in the soma and the germ line and strongly reduces mature miR-7 levels in the female germ line, suggesting that Loqs function is required for miRNA-directed silencing in vivo. A miRNA-regulated yellow fluorescent protein (YFP) reporter was introduced into loqsf00791 homozygous mutant flies. This transgenic reporter expresses in the eye a YFP mRNA bearing four miR-277 binding sites in its 3' UTR. The four miRNA-binding sites pair with all but the central three nucleotides of miR-277 and are, therefore, predicted to repress reporter mRNA translation rather than trigger mRNA cleavage. YFP fluorescence was readily detected in the eye and antennae in control flies in which the 3' UTR of the YFP transgene lacked the four miR-277 binding sites. When the reporter contained the miR-277 binding sites, YFP expression was repressed in the eye but readily visible in the antennae, indicating that miR-277 is expressed in the eye. This expression was verified independently by Northern blots of RNA isolated from eyes dissected away from other tissues of the head. Silencing of the miR-277-responsive YFP reporter in the eye was reduced in loqsf00791 homozygous mutant flies. As a control, the effect of a strong r2d2 mutation on YFP reporter expression was examined. The maximum fluorescence intensity in each eye was measured for all four genotypes. There was a significant increase in YFP fluorescence in eyes homozygous for the weak hypomorphic allele loqsf00791. This allele reduced miR-277 levels in the soma approximately 2-fold; fluorescence in the eye of homozygous mutant loqs flies was 1.8 ± 0.17 times greater than in the eyes of their age-matched heterozygous siblings. In contrast, flies homozygous for a strong hypomorphic r2d2 mutation show only a modest change in fluorescence. The Dcr-2 partner protein R2D2 is required for RNAi triggered by exogenous dsRNA or transgenes expressing long dsRNA hairpins. It is concluded that the reduced levels of Loqs protein in the loqsf00791 mutant lead to a statistically significant reduction in miRNA-directed silencing and that the Loqs paralog R2D2 plays little, if any, role in miRNA function (Forstemann, 2005).

dsRNA transcribed as an inverted repeat (IR) triggers silencing of corresponding mRNAs in flies. For IR-silencing of the white gene, whose gene product is required to produce the red pigment that colors fly eyes, the extent of silencing is proportionate to the number of copies of the IR-white transgene, but is relatively insensitive to the number of copies of white present. RNAi in Drosophila requires both Dcr-2, which transforms long dsRNA into siRNA, and R2D2, which collaborates with Dcr-2 to load siRNA into RISC. Thus, IR-silencing of white mRNA is lost in both dcr-2 and r2d2 mutant flies. The extent of white silencing was quantified by extracting the eye pigment in acidic ethanol and measuring its absorbance at 480 nm. Loss of R2D2 function in flies expressing one (or two) copies of the white IR transgene and two copies of the endogenous white locus restores red pigment levels to 74% (or 73% for two copies of IR-white) of wild-type flies lacking the white-IR. loqsf00791 mutant flies are also defective in IR-triggered white silencing, but to a much smaller extent. The loqsf00791 mutation restores pigment levels in flies carrying one copy of the white IR-expressing transgene to 12% of wild-type and to 8% for flies carrying two copies of the white-IR. loqsf00791 heterozygotes were statistically indistinguishable from wild-type flies bearing one copy of IR-white, whose eye pigment concentration was 4% (or 2% for two copies of IR-white) wild-type in the absence of the IR-white transgene (Forstemann, 2005).

The loqsf00791 allele consists of an insertion of the mini-white-expressing piggyBac transposon. Thus, loqsf00791 heterozygotes have two copies of the endogenous white locus and one copy of mini-white; loqsf00791 homozygotes have two copies of endogenous white and two copies of mini-white. The presence of this additional copy of mini-white does not account for the darker red color of white-silenced loqsf00791 flies, because loqsf00791 heterozygotes bearing two copies of white, one copy of mini-white (in the piggyBac transposon inserted at loqs), and one copy of a P-element expressing mini-white are effectively silenced by IR-white. In the absence of the IR-white transgene, the total amount of white expression in these flies is higher than in loqsf00791 homozygotes. Thus, reduction of Loqs function accounts for the partial desilencing of white in this system. The modest loss of silencing in the loqsf00791 mutant flies may reflect the incomplete loss of Loqs protein in this allele. However, a dcr-1 null mutation leads to a similar, partial loss of white IR-silencing. The small eye phenotype of dcr-1 null mutants unfortunately renders a quantitative comparison to loqsf00791 impossible. It is proposed that -- as for pre-miRNA processing -- Dcr-1 and Loqs act together to enhance silencing by siRNAs (Forstemann, 2005).

loqsf00791 males are incompletely fertile. When Oregon R females were mated to loqsf00791 homozygous mutant males, only 17% of embryos hatched; for loqsf00791 heterozygous males, 47% of embryos hatched. Ninety percent of embryos hatched for wild-type Oregon R males. Genes required for RNA silencing often reduce male fertility, because the X-linked gene Ste is epigenetically silenced in testes by dsRNA derived from the bi-directionally transcribed Suppressor of Stellate (Su(Ste)) locus. Ste silencing is genetically similar, but not identical, to RNAi, in that like RNAi it requires the function of the gene armitage (armi), but unlike RNAi does not require r2d2. In the absence of Ste silencing, Stellate protein accumulates as protein crystals in the testes. loqsf00791 mutants contain Stellate crystals in their testes, much like armi72.1 mutants, identifying a second role for loqs in silencing by endogenous RNA triggers, distinct from its function in miRNA biogenesis (Forstemann, 2005).

The loqs gene has a critical function in oogenesis, since loqsf00791 females have small ovaries and are completely sterile. Drosophila ovaries comprise ovarioles that contain developmentally ordered egg chambers, which are produced continuously in the adult by germ-line stem cell division. As a result, mutations that block stem cell division or maintenance lead to ovarioles containing few egg chambers. loqsf00791 mutant females lay no eggs. Whereas wild-type females contain 7 ± 0.8 previtellogenic egg chambers per ovariole, loqsf00791 contain only 3 ± 0.8. Excision of the piggybac transposon in loqsf00791 restores fertility, demonstrating that these defects reflect loss of Loqs function. The mature oocytes in loqsf00791 ovarioles have normal dorsal appendages, indicating that dorsoventral patterning is normal. In contrast, mutations in armi, spnE, and aub disrupt both dorsoventral and anteroposterior patterning. These mutations all disrupt RNAi and Ste silencing, but display no global defects in miRNA biogenesis or function, unlike loqs (Forstemann, 2005).

Oogenesis is initiated in the germarium, which contains the germ-line stem cells as well as the early germ-line cysts that will form egg chambers. In loqsf00791 mutant ovarioles, the germaria generally contain a limited number of cells that stain for Vasa, indicating that they are of germ-line origin. No mitotic figures were observed, nor were separate cysts. Germ-line stem cells and their daughter cells, the cystoblasts, are characterized by the presence of a spherical structure, the spectrosome, that stains intensely with anti-Spectrin antibodies. Wild-type and loqsf00791 germaria were stained with anti-α-Spectrin antibodies. Spectrosomes could not be detected in the loqs mutant germaria, suggesting that in these germaria, dissected from flies 3-4 d old, no stem cells remained. Stem cells must have originally been present, because loqs mutant ovaries produce some late-stage oocytes. Thus, most of the original stem cells may have died or differentiated into cystoblasts without renewing the stem cell pool. At present, we cannot distinguish between these alternatives. It is concluded that loqsf00791 mutants, which are defective in three distinct types of RNA silencing, fail to maintain germ-line stem cells (Forstemann, 2005).

Collectively, Dcr-1 and Loqs, Drosha and Pasha, and Dcr-2 and R2D2 comprise six of the 12 dsRBD proteins predicted to be encoded by the Drosophila genome. Thus, at least half of all dsRBD proteins in flies participate in RNA silencing. In Caenorhabditis elegans, the R2D2-like dsRBD protein RDE-4 is required for RNA interference and interacts with DCR-1, the sole worm Dicer gene. RDE-4 is equally similar to Loqs (E-value = 0.03) and R2D2 (E-value = 0.026; search restricted to C. elegans proteins). The Drosha/Pasha complex is also present in C. elegans. Similarly, the Arabidopsis thaliana dsRBD protein HYL1 is required for the production of mature miRNAs, and hyl1 mutant plants have a phenotype similar to that of dicer-like 1 (dcl1). It has been recently demonstrated that HYL1 is a dsRNA-binding protein that binds DCL1 and that the HYL1 paralog DRB4 binds the Dicer protein DCL4. Pairing of RNase III endonucleases with dsRBD proteins is thus a recurring theme in RNA silencing (Forstemann, 2005).

The human genome encodes one Dicer protein, which is more closely related to Drosophila Dcr-1 than Dcr-2. Sequence analysis of human proteins for similarity to either C. elegans RDE-4 or Drosophila R2D2 does not identify a reasonable candidate for a dsRBD partner protein for human Dicer. In contrast, the human TRBP is highly similar to Drosophila Loqs (E-value = 5 × 10−36). For comparison, the human proteins most similar to R2D2 or RDE-4 give E-values of 8 × 10−8 and 0.42, respectively, when the search is restricted to human proteins. Human TRBP was first identified ( Gatignol, 1991) because it binds HIV trans-activator RNA (TAR), a stem-loop structure required for active HIV transcription. Remarkably, the secondary structure of TAR resembles a miRNA precursor, and the recent discovery of Epstein-Barr virus-encoded miRNAs has fueled speculation that TAR may be a viral pre-miRNA (Forstemann, 2005 and reference therein).

Deletion of PRBP, the mouse homolog of TRBP, yields viable mice that often die at the age of weaning. Surviving homozygous mutant males show defects in spermatogenesis attributed to abnormal sperm maturation rather than proliferation (Zhong, 1999). In contrast, Dicer knockout mice show very early embryonic lethality. If mouse Dicer and PRBP collaborate to produce mature miRNA, the essential function of Dicer during mouse development must either be independent of miRNA function, or a redundant factor must replace PRBP during embryonic development but not spermatogenesis (Forstemann, 2005).

Together with dcr-1, the gene loqs is required in flies for normal pre-miRNA processing. Loqs and Dcr-1 reciprocally co-immunoprecipate. Pre-miRNA processing activity also co-immunoprecipitates with Dcr-1 and Loqs. However, in gel filtration chromatography, the two proteins overlap but do not precisely co-purify. Loqs and Dcr-1 may form a protein complex analogous to the Dcr-2/R2D2 and Drosha/Pasha complexes, but this complex may be transient, with Loqs also associating with other components of the RNA silencing machinery, perhaps even escorting the mature miRNA to Ago1, an approximately 110-kDa Argonaute protein associated with mature miRNAs in flies. In fact, the predominant Loqs-containing complex in S2 cell lysate is about 150 kDa larger than the peak of Dcr-1, so it could contain Dcr-1, Loqs, and Ago1. The data of Siomi and co-workers demonstrating that Ago1 associates with both Dcr-1 and Loqs (Saito, 1995) support such a view (Forstemann, 2005).

In humans and C. elegans, a single Dicer gene is responsible for generating both siRNAs and miRNAs. Drosophila has apparently duplicated both its ancestral Dicer RNase III endonuclease and its dsRBD partner protein, dedicating Dcr-1/Loqs to miRNA processing and Dcr-2/R2D2 to RNAi. Nonetheless, these two pathways are not completely separate, because cells lackingdcr-1 are not fully competent for IR-triggered silencing. Dcr-1 is not required for siRNA production, yet embryo extracts lacking Dcr-1 fail to assemble RISC. Dcr-1 has been proposed to be a component of 'holo-RISC', an 80S complex containing many, but not all, components of the RNAi pathway in flies. The loqsf00791 mutation also reduced the efficiency of IR-triggered silencing in vivo. Therefore, it is proposed that Dcr-1 must partner with Loqs not only during the processing of pre-miRNA to mature miRNA, but also to ensure Dcr-1 function in the Dcr-2-dependent RNAi pathway (Forstemann, 2005).

No function for Dcr-2 in miRNA biogenesis has been found. Consistent with these results, this study found little if any requirement for R2D2 in miRNA-directed silencing. Moreover, null or strong hypomorphic alleles of either dcr-2 or r2d2 show no overt phenotype, whereas the dcr-1Q1147X null mutation is embryonic lethal (Forstemann, 2005).

Endogenous silencing of the Stellate locus in testes is genetically distinct from miRNA-directed silencing, because it requires armitage, a gene that plays no general role in miRNA biogenesis or function. Stellate silencing resembles RNAi in that Stellate expression is repressed by a dsRNA trigger transcribed from the Su(Ste) gene. Su(Ste) dsRNA produces siRNAs, called repeat-associated siRNAs, that are longer than the siRNAs produced in the RNAi pathway in Drosophila. Even the weak allele described in this study, loqsf00791, which reduces loqs mRNA levels only approximately 3-fold in testes, dramatically de-silences Stellate. Given the intimate association of Dcr-1 with Loqs, the data raise the possibility that Loqs acts to silence Stellate in collaboration with Dcr-1, which may generate the Su(Ste) repeat-associated siRNAs (Forstemann, 2005).

The loqsf00791 mutation is the first viable allele in Drosophila with a generalized defect in miRNA production. The allele may therefore be useful for future phenotypic analysis of miRNA-dependent pathways during the life cycle of Drosophila. The most obvious phenotype of loqsf00791 is female sterility. loqsf00791 homozygotes produce few egg chambers, indicating a defect in germ-line stem cell maintenance or division. The loqsf00791 phenotype is similar to mutants in piwi, which encodes a member of the Argonaute protein family of core RISC components. In piwi mutant ovaries, germ-line stem cells fail to divide and instead differentiate directly into cystoblasts, depleting the germarium of germ-line stem cells. loqs mutants display a similar phenotype: no germ-line stem cells (i.e., spectrosome-containing cells) were detected in loqsf00791 homozygous germaria, suggesting that Loqs is required to maintain stem cells. Piwi is required in terminal filament cells, somatic cells surrounding the tip of the germarium, to send a signal that prevents germ-line stem cells from differentiating. Piwi is also required in germ-line stem cells themselves to stimulate their proliferation. Perhaps Piwi is at the core of an effector complex loaded with small RNA produced by Dcr-1 and Loqs. Intriguingly, dcr-1 knockout mice die at embryonic day 7.5, apparently devoid of stem cells (Forstemann, 2005).

RAX is required for fly neuronal development and mouse embryogenesis

RAX was originally discovered as the unique cellular activator for the dsRNA-dependent, interferon-inducible protein kinase PKR. Recent findings indicate that RAX is also a critical component of the RNA-induced silencing complex and a regulator of transcription. This study reports novel phenotypes for both fruit flies carrying a transposon insertion in the 5' UTR of dRax (independently identified as loqs/R3D1) and mice with a deletion of the entire Rax gene. In Drosophila a high level of dRax expression was detected in the developing nerve cord. Mutant fly embryos homozygous for the insertion dRax[f00791] display highly abnormal commissural axon structure of the CNS and 70% of the flies homozygous for the mutant allele die prior to adulthood. Surviving male flies have reduced fertility and female flies are sterile. Furthermore, these flies appear to have a severe defect in nervous system coordination or neuromuscular function resulting in significantly reduced locomotion. Mice were also generated that are heterozygous for a deletion of the entire Rax gene (exons 1-8). While mice that are heterozygous for the mutant allele are viable and appear normal, it was not possible to obtain mice homozygous for this mutant allele. Furthermore, no embryos were obtained by mating heterozygous mice at either E3.5, 7, or 14, that is nullizygous for the Rax gene. Since Rax is expressed in preimplantation blastocysts, these data indicate that deletion of the entire Rax gene is embryonic lethal in mice at a preimplantation stage of development. Collectively, these findings in two different species illustrate the importance of RAX for embryonic development (Bennett, 2008).

dRax is required for proper nervous system development during embryogenesis. In mutant embryos homozygous for the insertion dRax[f00791], both the longitudinal and commissural axon structures of the CNS are highly abnormal, reflecting the disorganization of the nervous system. The mutant is a PBac insertion in the first exon of dRax directly upstream of the translation start site that creates a hypomorphic mutant allele by attenuating its transcription. In situ hybridization analysis of the dRax[f00791] mutant embryos demonstrates a significant decrease of dRax expression in the developing nerve cord. However, this is not a null mutant since there are detectable levels of dRax expression in the nerve cord of the mutant embryos. Nonetheless, about 70% of homozygous animals die before adulthood while surviving males have reduced fertility and females are sterile. The CNS abnormality observed for mutant embryos manifested at the end of embryogenesis. A general, albeit subtler, abnormality in the CNS could also be detected using markers of specific neurons, such as Futsch (22C10) and Elav. However, of the markers tested, BP102 staining that labels all axons in the CNS revealed the most striking phenotype. Interestingly, it was observed that the surviving homozygous flies are often stuck and unable to escape from food, which indicates a severe defect in nervous system coordination or neuromuscular function. Furthermore, using a Random Locomotion Test it was observed that male homozygous dRax mutants collected 3 days after eclosion have a 69% decrease in locomotor activity compared to heterozygous control flies. Taken together these results point to the requirement of dRax for nervous system development (Bennett, 2008).


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Reference names in red indicate recommended papers.

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loquacious : Biological Overview | Regulation | Developmental Biology | Effects of Mutation or RNAi

date revised: 28 December 2012

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