Methods
Cell culture, plasmid transfection, and generation of stable or
knockout cell line in BmN4 cells
BmN4 cells (provided by T. Kusakabe, Kyushu University; not authen-
ticated and not tested for mycoplasma contamination) were cultured
at 27 °C in IPL-41 medium (AppliChem) supplemented with 10% fetal
bovine serum. For plasmid transfection, 5−7.5 μg of plasmid DNAs
were transfected into BmN4 cells (2.5 × 10^6 cells per 10 cm dish) with
X-tremeGENE HP DNA Transfection Reagent (Sigma). For generation
of stable cells expressing GFP-BmArmi, BmN4 cells were transfected
with a GFP-tagged BmArmi expression vector and selected under 10
μg/ml puromycin for 3 weeks. For generation of Trimmer knockout
cell line, BmN4 cells were co-transfected with pIEx1-MychCas9NLS
expression vector and pBS-BmU6-sgTrimmer expression vector. One
week later, the cells were reseeded at a low density (~1–4 × 10^4 cells per
15 cm dish) and cultured in 50–75% conditioned medium. About 3 weeks
later, colonies were picked up under a microscope.
Plasmid construction
pIEx1-Trimmer WT and the catalytic mutant (E30A) were described
previously^3. The primer sequences for plasmid construction are listed
in Supplementary Table 1.
pIEx1-MychCas9NLS. A DNA fragment coding Myc-hCas9-NLS was am-
plified from pRB14 (a gift from K. Förstemann)^32 and cloned into pIEx-1
vector (Millipore/Novagen) by In-Fusion HD cloning kit (Takara Clontech).
pBS-BmU6-sgTrimmer. To generate pBS-BmU6-BbsI-chiRNA vector,
the fly U6 promoter in pBS-U6-BbsI-chiRNA expression vector (a gift
from K. Förstemann)^32 was replaced with the Bombyx mori U6 pro-
moter^33 amplified from the BmN4 genome. Synthesized DNA oligos
for Trimmer sgRNA were annealed and inserted into BbsI-digested
pBS-BmU6-BbsI-chiRNA vector.
pIExZ-BmZuc WT, H141N. To generate pIExZ vector, the ampicillin resistant
gene and its promoter sequence in pIEx-1 vector (Millipore/Novagen) were
replaced by ie2 promoter and Zeocin resistant gene amplified from pIZ/V5-
His vector (Thermo Fisher/Invitrogen). To enhance the expression, BmZuc
coding sequence was codon-optimized to Bombyx mori using EMBOSS
Backtranseq (http://www.ebi.ac.uk/Tools/st/emboss_backtranseq/). The
BmZuc coding sequence was synthesized by GeneArt Strings DNA Frag-
ments service (Life Technologies) and cloned into pIExZ vector. The cata-
lytic mutant BmZuc (H141N) was generated by site-directed mutagenesis.
EGFP-BmArmi K692A. The ATP-binding mutant EGFP-BmArmi K692A
was generated by site-directed mutagenesis into EGFP-BmArmi (a gift
from T. Kusakabe and T. Tatsuke)^34.
Antibodies and western blotting
Rabbit anti-Siwi, anti-BmAgo3, anti-BmZuc, anti-BmArmi, anti-BmG-
PAT1, anti-BmGasz antibodies were generated by immunizing N-ter-
minally His-tagged recombinant Siwi (aa 2−100), BmAgo3 (aa 2−100),
BmZuc (aa 28−206), BmArmi (aa 2−294), BmGPAT1 (aa 602−870),
BmGasz (aa 2−269) respectively (Scrum). The sera were affinity-puri-
fied by a column containing the immobilized recombinant protein.
Anti-Trimmer and anti-BmPapi antibodies were described previously^3.
Anti-Flag (M2) (Sigma), anti-actin (Santa Cruz, sc-1616) and anti-GFP
(B-2) (Santa Cruz) antibodies were purchased. Chemiluminescence
was induced by Luminata Forte Western HRP Substrate (Millipore)
and images were acquired by Amersham Imager 600 (GE Healthcare).
In vitro processing assay
In vitro ssRNA loading and trimming, NaIO 4 -mediated oxidation, and
β-elimination were performed essentially as described previously^24.
Each substrate ssRNA was 5′-radiolabelled with T4 polynucleotide
kinase (Takara) and [γ-^32 P]ATP (PerkinElemer). For BmZuc cleavage
assay, Tri-KO cells were resuspended in hypotonic buffer (10 mM
HEPES-KOH (pH7.4), 10 mM KCl, 1.5 mM MgCl 2 , 1 mM DTT, 1 × Complete
EDTA-free protease inhibitor (Roche)) and incubated on ice for 20 min.
Subsequently, the cell suspension was vortexed for 30 s, centrifuged
at 1,000g for 20 min at 4 °C, and the supernatant was removed. The
pellet was resuspended in hypotonic buffer and used as the 1,000g
pellet fraction. Typically, 7 μl of the resuspended 1,000g pellet fraction
was added to immunopurified Flag–Siwi–ssRNA complex on beads
together with 3 μl of 40 × reaction mix (containing ATP, ATP regen-
eration system, and RNase inhibitor)^35 and incubated at 25 °C for 2.5 h
(Fig. 3c, e and Extended Data Fig. 4b, c) or 30 °C for 20 min (Fig. 3k),
2 h (Fig. 4d and Extended Data Fig. 5c), 2.5 h (Fig. 3h, i), or 3 h (Fig. 3d and
Extended Data Fig. 4f ). For BmZuc cleavage assay in an ATP-depleted
condition (Extended Data Fig. 4b), hypotonic buffer was added instead
of 40 × reaction mix. For standard trimming assay, Flag–Siwi–ssRNA
complex on beads was incubated with 1,000g pellet from naive BmN4
cells together with 40× reaction mix at 25 °C for 20 min (Extended Data
Fig. 2f ) or 30 °C for 1.5 h (Fig. 3i). In all the in vitro cleavage/trimming
assays, lysates with an equal protein concentration were used in each
experimental set. Images were acquired by Typhoon FLA 7000 (GE
Healthcare) and analysed using Multi Gauge 3.0 (Fujifilm).RNAi in BmN4 cells
For dsRNA preparation, template DNAs were prepared by PCR using
primers containing T7 promoter listed in Supplementary Table 1.
dsRNAs were transcribed using T7 Scribe Standard RNA IVT Kit (Cell
Script) and purified with MEGAclear Transcription Clean-Up Kit
(Thermo Fisher/Invitrogen). For dsRNA transfection, 5 μg of dsRNAs
were transfected into BmN4 cells (6 × 10^5 cells per 10 cm dish) with
X-tremeGENE HP DNA Transfection Reagent (Sigma). dsRNAs were
repeatedly transfected every 3 days for four times.Immunoprecipitation
For Siwi and BmAgo3 immunoprecipitation, cells were resuspended
in buffer A (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1.5 mM MgCl 2 , 0.2%
sodium deoxycholate, 0.1% lithium dodecyl sulfate, 0.4% NP-40, 0.5 mM
DTT, 1× Complete EDTA-free protease inhibitor (Roche)) and incubated
on ice for 20 min. The cell suspension was diluted with equal volume
of buffer A without detergents and centrifuged at 17,000g for 30 min
at 4 °C. The supernatant was incubated with normal rabbit IgG (Cell
Signaling), anti-Siwi or anti-BmAgo3 antibody at 4 °C for 1 h, and then
Dynabeads Protein G (Thermo Fisher/Invitrogen) was added. After
incubation at 4 °C for 1 h, the beads were washed with buffer B (25 mM
Tris-HCl (pH 7.6), 150 mM NaCl, 1.5 mM MgCl 2 , 0.1% sodium deoxycho-
late, 0.05% lithium dodecyl sulfate, 0.2% NP-40, 0.5 mM DTT, 1 × Com-
plete EDTA-free protease inhibitor (Roche)).The immunoprecipitated
proteins were eluted with SDS sample buffer, and bound RNAs were
purified with mirVana miRNA Isolation Kit (Thermo Fisher/Invitrogen)
for small RNA library preparation or TRI Reagent (Molecular Research
Center) for 5′ radiolabelling.Genome extraction, RNA extraction, quantitative real-time PCR
and northern blotting
Genomic DNA of BmN4 cells was extracted using NucleoSpin Tissue
(Macherey-Nagel). Total RNAs prepared by TRI Reagent (Molecular
Research Center) were used for real-time PCR and northern blotting.
One microgram of total RNAs was reverse transcribed by PrimeScript
RT reagent kit with gDNA eraser (Takara), and qRT–PCR was performed
using KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) and the
Thermal Cycler Dice Real Time System (Takara). For northern blot-
ting, 10−12 μg of total RNAs were resolved by 15% urea polyacrylamide
gel electrophoresis (PAGE) and transferred to Hybond-N membrane
(GE Healthcare). After chemical crosslinking^36 , 5′ labelled antisense