Construction of reporter plasmids and RNAi trigger dsRNAs
To create an expression vector for both cultured cells and transgenic flies, we PCR amplified the 3′ UTR and SV40 poly-A signal from plasmid pEGFP-N1 (Clontech, Mountain View, CA, USA) with oligonucleotides 5′-ATC ACT CTC GGC ATG GAC GAG-3′ and 5′-GTG AAT TCA TAC ATT GAT GAG TTT GGA C-3′ and inserted the resulting PCR product into pUbi-Casper2 (a kind gift of Dr. Siu Ing The) using the NotI and EcoRI restriction sites, creating vector pKF60. For the GFP-insert, we transferred a BamHI-NotI fragment from pEGFP-N1 (Clontech) into pBluescript (Stratagene, La Jolla, CA, USA) cut with BamHI/NotI, creating pKF20. Subsequently, we annealed the oligos 5′-CAT GGA ACA AAA ACT TAT TTC TGA AGA AGA CTT GGG-3′ and 5′-CAT GCC CAA GTC TTC TTC AGA AAT AAG TTT TTG TTC -3′, encoding a myc-tag, and ligated this DNA-fragment into NcoI-cut pKF20. After sequencing, one clone was selected that contained a triple insertion in the correct orientation (pKF30). From this plasmid, the myc3-GFP-sequence was transferred as a BamHI-NotI-fragment into pKF60, resulting in plasmid pKF62. To remove an XbaI-site from the pCASPER2 polylinker, pKF62 was cut with XbaI, the ends treated with Klenow polymerase (New England Biolabs, Ipswitch, MA, USA), and the vector was re-ligated, creating pKF63. This plasmid was transformed into dam/dcm negative bacteria (strain GM2163, New England Biolabs), which rendered a second XbaI-site in the 3′-UTR, adjacent to the NotI-site, cleavable. To insert the miR-277 target sites, we annealed oligos 5′-GGC CTG TCG TAC CAG ATA GTG CAT TTA CAG TGT CGT ACC AGA TAG TGC ATT TA-3′ and 5′-CTA GTA AAT GCA CTA TCT GGT ACG ACA CTG TAA ATG CAC TAT CTG GTA CAG CA-3′ for the two perfectly matched sites, and oligos 5′-GGC CTG TCG TAC CAG AGG ATG CAT TTA CAG TGT CGT ACC AGA GGA TGC ATT TAT GTC GTA CCA GAG GAT GCA TTT ACA GTG TCG TAC CAG AGG ATG CAT TTA -′ and 5′-CTA GTA AAT GCA TCC TCT GGT ACG ACA CTG TAA ATG CAT CCT CTG GTA CGA CAT AAA TGC ATC CTC TGG TAC GAC ACT GTA AAT GCA TCC TCT GGT ACG ACA-3′ for the four bulged sites, then ligated the DNA fragments into NotI-XbaI-cut pKF63, creating pKF67 and pKF68, respectively.
These pCASPER2-derived expression plasmids were used both for the generation of stable S2-cell lines and for the P-element-mediated genetic transformation Drosophila melanogaster
(Rubin and Spradling, 1982
Constructs to make dsRNA directed against GFP, dcr-1
were described previously (Forstemann et al., 2005
). Templates for the synthesis of dsRNA directed against ago1
were generated by T/A-cloning PCR products generated using the oligonucleotides 5′-CGC ACC ATT GTG CAT CCT AAC GAG-3′and 5′-GGG GAC AAT CGT TCG CTT TGC GTA-3′ for ago2
and 5′-ATT TGA TTT CTA TCT ATG CAG CCA-3′ and 5′-GCC CTG GCC ATG GCA CCT GGC GTA-3′ for ago1
into the modified Litmus28i vector described previously (Forstemann et al., 2005
). The template for producing dsRNA targeting r2d2
was generated by PCR using oligonucleotides 5′-CGT AAT ACG ACT CAC TAT AGG CAT ACA CGG CTT GAT GAA GGA TTC-3′ and 5′-CGT AAT ACG ACT CAC TAT AGG TTG CTT GTG CTC GCT ACT TGC-3′. Templates for in vitro transcription were generated by PCR-amplification of each plasmid construct using a single primer corresponding to the T7 promotor (5′-CGT AAT ACG ACT CAC TAT AGG-3′) and dsRNA for knock-down was generated as described in (Haley et al., 2003
Construction of cell lines with increased miR-277 expression
A 270 nt fragment of genomic DNA surrounding the miR-277 sequence was PCR-amplified from S2-cell genomic DNA with the oligonucleotides 5′-GCG GAT CCG GTA CCT ATA CAT ATA TAA CGA GGC CTA ACG-3′ and 5′-ATG CGG CCG CAA AAC AGT GTC TTA CAA ACA AGT GG-3′. The resulting DNA fragment was cloned BamHI to NotI into pKF62, yielding mini-pri-miR-277 transgene under the control of the ubiquitin promotor. miR-277 over expression was quantified by comparing the amount of miR-277, relative to the amount of endogenous bantam miRNA, in extracts from cells expressing only endogenous miR-277 and in cells containing the mini-pri-miR-277 transgene ().
Cell culture and flow cytometry
Drosophila Schneider 2 cells were cultured at 28°C in Schneider's medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen). GFP expression plasmids were transfected with siLentfect (see below) at 1 μg of plasmid per well of a 24-well plate. For selection of stable transformants, 20 ng of phsNeo (Steller and Pirrotta, 1985
) was co-transfected with 1 μg each GFP reporter plasmid. Five days after transfection, the cells were split 1:5 into medium supplemented with 1.2 mg/ml G418 (Invitrogen). The cells were diluted 1:5 every 7 days into G418-containing medium for three weeks, then serial dilutions were plated into a 96-well plate in growth medium supplemented with 1% (v/v) sterile-filtered medium used previously for S2-cell culture (conditioned medium). After two weeks of growth, wells with a single colony of cells were expanded and analyzed by flow cytometry. Cell clones that produced a single peak in the flow cytometer were retained for use as reporter cells.
For transfection with RNAi-triggers, the cells were seeded at a density of 1 × 106
cells/ml in 24 well plates (500 μl/well) using Schneider's medium without G418. Liposome/nucleic acid complexes were prepared with 1.5 ml siLentfect (BioRAD, Hercules/CA, USA) and either 1 μg of dsRNA (estimated by native agarose gel electrophoresis) or 10 pmol of cholesterol conjugated, 2′-O
-methyl modified antisense-oligonucleotide (see Figure S1B
) per well in a total volume of 100 μl Schneider's medium without serum, incubated at room temperature for 45 min and added to the cells (100 μl/well of a 24 well plate). After 6 days, the cells were analyzed by FACS (BD FACScan flow cytometer; Becton Dickinson, Franklin Lakes, NJ). GFP-expression was quantified by determining the arithmetic mean of the fluorescence using the CellQuest software package (Becton Dickinson).
Generation of anti-Dcr-1 and anti-Ago2 polyclonal antibodies
The KLH-conjugated peptide CQGLIAKKD was used to immunize rabbits to generate anti-Dcr-1 antibodies (Covance Research Products, Denver, PA, USA) as described (Bernstein et al., 2001
). The antiserum was affinity-purified using the synthetic peptide coupled to a column matrix (Sulfolink, Pierce, Rockford, IL, USA) via an N-terminal cysteine according to the manufacturer's protocol. To generate anti-Ago2 antibodies, rabbits were immunized (ProSci, Poway, CA, USA) with the Ago2 PAZ-domain fused to glutathione-S-transferase expressed and purified as described (Lingel et al., 2003
). The antiserum was purified using a NusA-Ago2-PAZ fusion protein (Lingel et al., 2003
) immobilized on a column matrix (Aminolink plus, Pierce) according to the manufacturer's protocol. The KLH-conjugated peptide CSDEYESSKDKAMD was used to immunize chickens to generate anti-R2D2 antibodies (Gallus Immunotech, Cary, NC, USA).
Proteins were extracted either from cultured S2 cells or from hand-dissected adult fly head and thorax with PBS containing 1% Triton X-100 (Sigma, St. Louis, MO, USA) and protease inhibitors (Complete without EDTA, Roche Molecular Biochemicals, Basel, Switzerland). For quantification of myc3GFP-expression in transgenic flies, 20 μg of total protein was resolved by electrophoresis through a 12% polyacrylamide/SDS gel and transferred to PVDF-membrane (Immobilon-P, Millipore, Billerica, MA, USA) by semi-dry transfer (BioRAD, Hercules, CA, USA) in 25 mM Tris (pH 8.3), 250 mM glycine, 10% (v/v) methanol as anode buffer and 20 mM CAPS, pH 11.0, as cathode buffer at 20 V for 120 min. Purified monoclonal anti-myc 9E10 (Sigma #M4439) was used diluted 1:1000 in 25 mM Tris, 137.5 mM NaCl, 2.5 mM KCl, 0.02% (v/v) Tween-20 (Sigma) for 90 min at room temperature. HRP-conjugated goat anti-rabbit secondary antibody, used at 1:1,000 dilution, and chemiluminescent substrate were from the Pierce SuperSignal West Dura kit (Pierce). HRP-conjugated rabbit anti-chicken secondary antibody (Gallus Immunotech), diluted 1:15,000, was used to detect the affinity purified anti-R2D2 chicken IgY. Western Blot images were acquired using a Fuji LAS-3000 (Fujifilm Life Sciences, Stamford, CT, USA) and quantified using ImageGauge (Fujifilm Life Sciences). As a loading control, α-tubulin was detected with anti-α-tubulin DM1A (Sigma #T6199) diluted 1:1000. A standard curve for myc3-GFP detection was created by diluting extract from pKF63-transgenic flies into extract from yw flies, and initial Western blot signals corrected using this standard curve.
To detect S2-cell proteins, 50 μg total protein was resolved by electrophoresis through an 8% polyacrylamide/SDS gel and transferred to PVDF as above. Incubation with primary antibodies was overnight at 4°C; secondary antibodies were incubated with the membrane for 120 min at room temperature. The primary antibodies were diluted 1:2000. Ant-Dcr-2 antibody was the kind gift of Qinghua Liu (Liu et al., 2003
Ago1 and Ago2 target cleavage kinetics
Target cleavage reactions were performed essentially as previously described (Haley and Zamore, 2004
; Haley et al., 2003
). In , 50 nM let-7
siRNA or let-7/let-7*
duplex was incubated with Drosophila
0–2 h embryo lysate for 2 min or 5 min to program Ago2-RISC (~4.7 nM) or Ago1-RISC (~4.6 nM), respectively. In , 20 nM let-7
siRNA or let-7/let-7*
duplex was incubated with lysate for 3 min or 8 min to program Ago2-RISC or Ago1-RISC, respectively. For Ago2 cleavage in , RISC was diluted 10-fold in N
-ethyl maleimide (NEM) treated embryo lysate (Haley and Zamore, 2004
; Nykanen et al., 2001
). Control experiments demonstrated that in ago2414
lysate the let-7
siRNA assembled little or no active RISC, as assessed by target cleavage activity, whereas let-7/let-7*
was as active in ago2414
as in wild-type lysate, indicating that the let-7
siRNA and let-7/let-7*
duplex are almost exclusively loaded Ago2- and Ago1-RISC, respectively.
RISC assembly was stopped by treatment with NEM followed by DTT to quench unreacted NEM for both Ago1- (Figure S4A
) and Ago2-RISC (Nykanen et al., 2001
). Control experiments (Figure S4B
) established that the biphasic kinetics of Ago1-RISC in the presence of ATP were not a consequence of treatment with NEM. RISC concentration was estimated by 2′-O
-methyl ASO affinity purification in and by the size of the pre-steady-state burst in (Haley and Zamore, 2004
; Schwarz et al., 2003
). The concentration of RNA target was 100 nM in and 0.5 to 100 nM in . Data were analyzed using IGOR 5 (WaveMetrics) and Visual
Enzymics 2005 (Softzymics) software.