Loqs, a dsRBD Partner Protein for <i>Drosophila</i> Dcr-1

<div><p>(A) Each of the three <i>D.</i><i>melanogaster</i> RNase III endonucleases pairs with a different dsRBD protein, which assists in its function in RNA silencing.</p> <p>(B) Differential splicing creates three <i>loqs</i> mRNA variants, <i>loqs</i> RA, RB, and RC. <i>loqs</i> RA and RB are reported in FlyBase. The RC splice variant is reported here. Arrows mark the position of the PCR primers used in (D); green lines, start codons; red lines, stop codons. The resulting protein isoforms are diagrammed to the right.</p> <p>(C) Use of an alternative splice acceptor site extends the 5′ end of exon 4. The mRNA sequence surrounding the new exon–exon junction is shown, with the <i>loqs</i> RC-specific sequence in bold; the arrow marks the position of the last nucleotide of exon 3 relative to the putative transcription start site. When translated into protein, the exon 4 extension inserts 43 new amino acids (indicated below the mRNA sequence) and shifts the Loqs PC reading frame, truncating the protein.</p> <p>(D) RT-PCR analysis of <i>loqs</i> mRNA species in males, female carcasses remaining after ovary dissection, dissected ovaries, and S2 cells. Males express more <i>loqs</i> RA than <i>loqs</i> RB, female somatic tissue expresses both <i>loqs</i> RA and <i>loqs</i> RB, while ovaries express predominantly <i>loqs</i> RB. <i>loqs</i> RC was observed only in S2 cells, together with <i>loqs</i> RA and <i>loqs</i> RB.</p> <p>(E) The piggyBac transposon insertion f00791 lies 57 bp upstream of the reported transcription start site for <i>loqs</i>.</p></div

Analysis of Complexes Containing Pre-miRNA Processing Activity, Dcr-1, and Loqs

<div><p>(A) S2 cell lysate was fractionated by gel filtration chromatography and analyzed for pre-<i>let-7</i> processing activity, and Dcr-1, Dcr-2, and Loqs proteins.</p> <p>(B) The sizes of the distinct complexes containing Loqs (∼630 kDa), Dcr-1 (∼480 kDa), and Dcr-2 (∼230 kDa) and the broad complex containing pre-miRNA processing activity (∼525 kDa) were estimated using molecular weight standards (thyroglobulin, 669 kDa; ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa; bovine serum albumin, 67 kDa; ovalbumin, 43 kDa; chymotrypsinogen A, 25 kDa) and recombinant Dcr-2 and R2D2 proteins (rDcr-2 and rR2D2). The blue asterisk denotes the peak of pre-<i>let-7</i> processing activity detected in (A).</p> <p>(C) Fractions containing the Dcr-1 peak were pooled and immunoprecipitated with either anti-Dcr-1 or anti-Loqs antibodies. Western blotting with anti-Dcr-1 and anti-Loqs antibodies demonstrated that Dcr-1 and Loqs remained associated through gel filtration chromatography.</p></div

Silencing of a miRNA-Responsive YFP Reporter Requires <i>loqs</i> but Not <i>r2d2</i>

<div><p>(A) A YFP transgene expressed from the Pax6-promoter showed strong fluorescence in the eye and weaker fluorescence in the antennae. Due to the underlying normal red eye pigment, the YFP fluorescence was observed in only those ommatidia that are aligned with the optical axis of the stereomicroscope. In heterozygous <i>loqs</i>^f00791/CyO flies bearing a miR-277-responsive, Pax6-promotor-driven, YFP transgene, YFP fluorescence was visible in the antennae but was repressed in the eye. In contrast, in homozygous mutant <i>loqs</i>^f00791 flies, YFP fluorescence was readily detected in the eye. A strong mutation in <i>r2d2</i> did not comparably alter repression of the miR-277-regulated YFP reporter. The exposure time for the unregulated YFP reporter strain was one-fourth that used for the miR-277-responsive YFP strain. The exposure times were identical for the heterozygous and homozygous <i>loqs</i> and <i>r2d2</i> flies.</p> <p>(B) Additional images of eyes from <i>loqs</i>^f00791 heterozygous and homozygous flies bearing the miR-277-responsive YFP reporter transgene diagrammed in (A).</p> <p>(C) Quantification of fluorescence of the miR-277-responsive YFP transgene in eyes heterozygous or homozygous for <i>loqs</i> or <i>r2d2</i>. The maximum pixel intensity was measured for each eye (excluding antennae and other tissues where miR-277 does not appear to function). The graph displays the average (<i>n</i> = 13) maximum pixel intensity ± standard deviation for each homozygous genotype, normalized to the average value for the corresponding heterozygotes. Statistical significance was estimated using a two-sample Student's <i>t</i>-test assuming unequal variance.</p> <p>The images in (A) were acquired using a sensitive, GFP long-pass filter set that transmits yellow and red autofluorescence. Images in (B) and for quantitative analysis were acquired using a YFP-specific band-pass filter set that reduced the autofluorescence recorded.</p></div

Loqs Is Associated with Pre-miRNA Processing Activity in S2 Cells

<div><p>(A) Pre-miRNA processing activity co-immunoprecipitates with myc-tagged Loqs PB and with endogenous Dcr-1 or endogenous Loqs, but not with myc-tagged GFP.</p> <p>(B) Pre-miRNA processing activity co-purifies by immunoprecipitation with both Loqs protein isoforms that interact with Dcr-1, Loqs PA, and Loqs PB. The extracts used in (A) and (B) were independently prepared.</p></div

loqs^f00791 Fail to Maintain Germ-Line Stem Cells

<div><p>(A) Wild-type ovarioles contain a germarium and a developmentally ordered array of six to eight egg chambers, whereas <i>loqs</i>^f00791 mutant ovarioles contain a smaller than normal germarium, two or three pre-vitellogenic egg chambers, and a late-stage egg chamber. Wild-type and <i>loqs</i> ovarioles are shown at the same magnification.</p> <p>(B) In wild-type ovarioles, the germarium contains several newly formed germ-line cysts surrounded by somatic follicle cells. In contrast, <i>loqs</i>^f00791 mutant germaria contain few germ-line cells, which are not organized into distinct cysts. The follicle cell layer is also significantly reduced in<i>loqs</i>^f00791 germaria.</p> <p>(C) Wild-type and <i>loqs</i> mutant germaria labeled for α-Spectrin (green) and filamentous Actin (red). In wild type, anti-α-Spectrin labels the spectrosome (ss), a structure characteristic of germ-line stem cells, which are normally found at the anterior of the germarium, apposed to the somatic terminal cells (tc). The cystoblasts, the daughters of the stem cells, also contain a spectrosome, but are located posterior to the stem cells. In <i>loqs</i> mutant ovaries, spectrosome-containing cells were not detected, indicating that normal germ-line stem cells are not present. These observations indicate that stem cells are not maintained.</p> <p>In (A) and (B), ovaries were labeled for filamentous actin (red) using rhodamine phalloidin, DNA (blue) using TOTO3 (Molecular Probes), and the germ-line marker Vasa (green) using rabbit anti-Vasa antibody detected with fluorescein-conjugated anti-rabbit secondary antibody. In (B) and (C), wild-type and <i>loqs</i> germaria are shown at the same magnification.</p></div

Silencing of <i>white</i> by an IR Partially Depends on <i>loqs</i>

<div><p>(A) The red eye color of wild-type flies (left) changes to orange (center) and white (right) in response to one or two copies, respectively, of a <i>white</i> IR transgene, which silences the endogenous <i>white</i> gene.</p> <p>(B) Homozygous mutant <i>r2d2</i> flies fail to silence <i>white,</i> even in the presence of two copies of the <i>white</i>-IR transgene; heterozygous <i>r2d2</i>/CyO flies repress <i>white</i> expression.</p> <p>(C) In flies homozygous for <i>loqs</i>^f00791, silencing of <i>white</i> by the <i>white</i>-IR is less efficient; two copies of the <i>white</i>-IR do not produce completely white eyes, whereas they do in heterozygous <i>loqs</i>^f00791/CyO.</p> <p>(D) The eye color change in <i>loqs</i>^f00791 flies is not caused by the increased <i>white⁺</i> gene dose resulting from the mini<i>-white</i> marker in the <i>piggyBac</i> transposon that causes the <i>loqs</i>^f00791 mutation. Flies <i>trans</i>-heterozygous for <i>loqs</i>^f00791 and a mini-<i>white</i>-marked P-element have more red eye pigment than <i>loqs</i>^f00791 homozygous flies, but show more efficient silencing by the <i>white</i>-IR than <i>loqs</i>^f00791 homozygous animals.</p> <p>(E) The eye pigment of the indicated genotypes was extracted and quantified by green light (480 nm) absorbance, relative to wild-type flies bearing no <i>white</i>-IR transgenes. The graph shows the mean and standard deviation of five independent measurements per genotype.</p></div

Loqs Is Required for Efficient pre-<i>let</i>-<i>7</i> Processing In Vitro

<div><p>(A) <i>loqs</i>^f00791 mutant ovary lysates processed pre-<i>let-7</i> into mature <i>let-7</i> miRNA ∼19-fold more slowly than wild-type. The data were fit to a first-order exponential equation, and initial velocities calculated from the fitted curve.</p> <p>(B) Analysis of pre-<i>let-7</i> processing in extracts from S2 cells. The cells were treated twice with dsRNA corresponding to the indicated genes.</p></div

Silencing of <i>Stellate</i> by the dsRNA-Generator <i>Su(Ste)</i> Requires <i>loqs</i>

<p>Testes were stained for DNA (red) and Stellate protein (green). Defects in RNA silencing often lead to accumulation of Stellate protein crystals in testes. For example, the testes from the strong allele <i>armi^72.1,</i> but not wild-type Oregon R testes, show Stellate protein staining. Testes from <i>loqs</i>^f00791 males show strong accumulation of Stellate protein, consistent with their significantly impaired fertility.</p