A 2′-<i>O</i>-Methyl Oligonucleotide Is a Potent Inhibitor of RNAi in Human Cultured HeLa Cells

<div><p>(A–D) HeLa cells were transfected with 1 nM (A), 5 nM (B), 10 nM (C), or 25 nM (D) siRNA-targeting <i>Pp-</i>luc mRNA. The next day the cells were cotransfected with <i>Rr-</i>luc<i>-</i>and <i>Pp</i>-luc-expressing plasmids together with various amounts of a 31-nt 2′<i>-O-</i>methyl oligonucleotide complementary to the antisense strand of the siRNA. The half-maximal concentration of 2′-<i>O</i>-methyl oligonucleotide required to inhibit (IC₅₀) was determined by fitting the data to a sigmoidal curve using a Hill coefficient of 1.</p> <p>(E) IC₅₀ plotted as a function of the concentration of transfected siRNA.</p></div

A 2′<i>-O-</i>Methyl RNA Oligonucleotide Inhibits RNAi In Vitro in <i>Drosophila</i> Embryo Lysate

<div><p>(A) Sequences of the sense and antisense <i>Pp</i>-luc target RNAs (black), the siRNA (red, antisense strand; black, sense strand), and the sense and antisense 2′<i>-O-</i>methyl oligonucleotides (blue) used.</p> <p>(B) Sequence-specific depletion of RNAi activity by immobilized 2′-<i>O</i>-methyl oligonucleotides from <i>Drosophila</i> embryo lysate programmed with siRNA. siRNA was incubated with lysate to assemble RISC; then, immobilized 2′-<i>O</i>-methyl oligonucleotide was added. Finally, the beads were removed from the supernatant, and either sense or antisense ³²P-radiolabeled target RNA was added to the supernatant to measure RISC activity for each siRNA strand.</p> <p>Symbols and abbreviations: Ø, target RNA before incubation with siRNA-programmed lysate; T, total reaction before depletion; unbound, the supernatant after incubation with the immobilized antisense (AS) or sense (S) 2′-<i>O</i>-methyl oligonucleotides shown in (A). The absence of 5′ cleavage product demonstrates that the sense oligonucleotide depleted RISC containing antisense siRNA, but not sense siRNA, and the antisense oligonucleotide depleted the sense RISC, but not that containing antisense siRNA. Bi, 5′ biotin attached via a six-carbon linker.</p></div

Injection of a 2′-<i>O</i>-Methyl Oligonucleotide Complementary to <i>let-7</i> miRNA Can Phenocopy the Loss of <i>let-7</i> Function in C. elegans

<div><p>(A) Wild-type and <i>lin-41(ma104)</i> L2-stage C. elegans larvae were injected with either a 2′<i>-O-</i>methyl oligonucleotide complementary to <i>let-7</i> miRNA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020098#pbio-0020098-g005" target="_blank">Figure 5</a>A) or an unrelated <i>Pp</i>-luc 2′-<i>O</i>-methyl oligonucleotide. Absence of alae and presence of bursting vulvae were scored when the injected animals reached adulthood.</p> <p>(B) Isolation of <i>let-7</i>-associated proteins with a tethered 2′-<i>O</i>-methyl oligonucleotide. Northern blot analysis of <i>let-7</i> miRNA remaining in the supernatant of the worm lysate after incubation with the <i>let-7</i>-complementary (<i>let-7</i>) or <i>Pp</i>-luc (unrelated) oligonucleotide. Input represents the equivalent of 50% of the total extract incubated with tethered oligonucleotide.</p> <p>(C) Western blot analysis of the GFP-tagged ALG-1 and ALG-2 proteins associated with <i>let-7</i>. The upper band corresponds to GFP-tagged ALG-1 and the lower to GFP-tagged ALG-2. Extracts from a transgenic strain expressing the tagged proteins was incubated with the indicated tethered 2′-<i>O</i>-methyl oligonucleotide; then, the beads were washed and bound proteins were fractionated on an 8% SDS-polyacrylamide gel. Western blots were probed using anti-GFP monoclonal or anti-RDE-4 polyclonal antibody. The RDE-4-specific band is marked with an asterisk (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020098#pbio-0020098-Tabara2" target="_blank">Tabara et al. 2002</a>).</p> <p>(D and E) Analysis of <i>let-7</i> miRNA in ALG-1/ALG-2 complexes (D). Extracts prepared from mixed-stage wild-type worms (N2) or from GFP::ALG-1/ALG-2 transgenic worms were immunoprecipitated using anti-GFP monoclonal antibodies. The unbound and immunoprecipitated RNAs were analyzed by Northern blot hybridization for <i>let-7</i> (D), and 5% of the immunoprecipitated protein was analyzed by Western blotting for GFP to confirm recovery of the GFP-tagged ALG-1/ALG-2 proteins (E).</p></div

A Complementary 2′<i>-O-</i>Methyl Oligonucleotide Blocks Endogenous <i>let-7</i>-Containing RISC Function

<div><p>(A) Sequence of the <i>let-7</i>-complementary site in the target RNA (black), of the siRNA (red, antisense strand; black, sense strand), and of the <i>let-7</i>-complementary 2′<i>-O-</i>methyl oligonucleotide (blue).</p> <p>(B) Schematic representation of the target RNA, which contained both <i>Pp</i>-luc and antisense <i>let-7</i> sequences.</p> <p>(C) <i>Drosophila</i> embryo lysate (left) was programmed with <i>let-7</i> siRNA; then, the target RNA and the 2′<i>-O-</i>methyl oligonucleotide were added together. Target RNA and 2′-<i>O</i>-methyl oligonucleotide (right) were added to HeLa S100 extract, which contains endogenous human <i>let-7</i>-programmed RISC.</p> <p>(D) An RNA target containing both <i>Pp</i>-luc and antisense <i>let-7</i> sequence can be simultaneously targeted by <i>Pp</i>-luc siRNA and endogenous <i>let-7</i> in HeLa S100 lysate. The <i>let-7</i>-complementary 2′-<i>O</i>-methyl oligonucleotide blocks <i>let-7</i>-programmed, but not <i>Pp</i>-luc siRNA-programmed, RISC function. The bottom panel shows the same samples analyzed separately to better resolve the <i>let-7</i> 5′ cleavage product.</p> <p>(E) <i>Drosophila</i> embryo lysate was programmed with <i>let-7</i> siRNA and then incubated with biotinylated 2′-<i>O</i>-methyl oligonucleotide tethered to paramagnetic streptavidin beads. The beads were removed and the supernatant tested for RNAi activity.</p> <p>Symbols and abbreviations: Ø, target RNA before incubation with siRNA-programmed lysate; T, total reaction before depletion; unbound, the supernatant after incubation with the paramagnetic beads. “Mock” indicates that no oligonucleotide was used on the beads; “<i>let-7</i>” indicates that the beads contained the <i>let-7</i>-complementary oligonucleotide shown in (A).</p></div

RISC Does Not Act through an Antisense Mechanism

<div><p>(A) Inhibition of sense target cleavage by an antisense 2′<i>-O-</i>methyl oligonucleotide requires an approximately 40-fold higher concentration than by a sense oligonucleotide. The antisense oligonucleotide can pair completely with the sense target RNA, but not with the antisense siRNA-programmed RISC. The IC₅₀ value and the RISC concentration are indicated. Also shown are the sequences of the sense <i>Pp</i>-luc RNA target (black), the siRNA (red, antisense strand; black, sense strand), and the 2′<i>-O-</i>methyl oligonucleotide (blue).</p> <p>(B) The same antisense 2′-<i>O</i>-methyl oligonucleotide is an effective competitor of antisense target cleavage. In this experiment, inhibition occurs via binding of the antisense oligonucleotide to the sense siRNA-programmed RISC, not the target RNA. The IC₅₀ value and the RISC concentration are indicated. Also shown are the sequences of the <i>Pp</i>-luc antisense RNA target (black), the siRNA (red, antisense strand; black, sense strand), and the 2′<i>-O-</i>methyl oligonucleotide (blue). The G:U wobble in the siRNA duplex in (B) acts to direct the sense strand into RISC and improving its efficacy in target cleavage.</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

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^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

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

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