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Supplementary information
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Supplementary materials and methods
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Preparation of mRNAs encoding TPR proteins for in vitro translation
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A DNA fragment encoding N-terminally myc-tagged FKBP62 was amplified by
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PCR using primer231 and primer232 (Supplementary Table S1) from the cDNA
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that was synthesized from A. thaliana (Col-0) total RNA using SuperScript III
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Reverse Transcriptase (Invitrogen, US) and primer230, digested with PstI and
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BamHI, and cloned into the pSP64-poly(A) vector to obtain pSP-mycFKBP62. To
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obtain pSP-mycFKBP65, a DNA fragment encoding N-terminally myc-tagged
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FKBP65 was amplified using primer260 and primer262 from the cDNA that was
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synthesized from A. thaliana (Col-0) total RNA with primer261, digested with SalI
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and
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pSP-mycCYP40, a DNA fragment encoding N-terminally myc-tagged CYP40
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was amplified using primer250 and primer252 from the cDNA that was
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synthesized from A. thaliana (Col-0) total RNA with primer251, digested with PstI
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and BamHI, and cloned into the pSP64-poly(A) vector. To obtain pSP-mycPP5, a
BamHI,
and
cloned
into
the
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pSP64-poly(A)
vector.
To
obtain
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DNA fragment encoding N-terminally myc-tagged PP5 was amplified using
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primer246 and primer248 from the cDNA that was synthesized from A. thaliana
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(Col-0) total RNA with primer247, digested with PstI and XbaI, and cloned into
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the pSP64-poly(A) vector. To obtain pSP-mycTPR1, a DNA fragment encoding
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N-terminally myc-tagged TPR1 was amplified using primer276 and primer278
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from the cDNA that was synthesized from A. thaliana (Col-0) total RNA with
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primer277, digested with PstI and BamHI, and cloned into the pSP64-poly(A)
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vector. To obtain pSP-mycTPR2, a DNA fragment encoding N-terminally
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myc-tagged TPR2 was amplified using primer293 and primer295 from the cDNA
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synthesized from A. thaliana (Col-0) total RNA with primer294, digested with PstI
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and BamHI, and cloned into the pSP64-poly(A) vector. To obtain pSP-mycTPR7,
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a DNA fragment encoding N-terminally myc-tagged TPR7 was amplified using
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primer285 and primer287 from the cDNA that was synthesized from A. thaliana
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(Col-0) total RNA with primer286, digested with PstI and XbaI, and cloned into
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the pSP64-poly(A) vector.
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To obtain pSP-FKBP62myc, a DNA fragment encoding C-terminally
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myc-tagged FKBP62 was amplified using primer306 and primer307 from
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pSP-mycFKBP62, digested with PstI and BamHI, and cloned into the
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pSP64-poly(A) vector. To obtain pSP-FKBP65myc, a DNA fragment encoding
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C-terminally myc-tagged FKBP65 fragment was amplified using primer308 and
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primer309 from pSP-mycFKBP65, digested with SalI and BamHI, and cloned
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into the pSP64-poly(A) vector. To obtain pSP-CYP40myc, a DNA fragment
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encoding C-terminally myc-tagged CYP40 was amplified using primer310 and
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primer311 from pSP-mycCYP40, digested with PstI and BamHI, and cloned into
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the pSP64-poly(A) vector. To obtain pSP-PP5myc, a DNA fragment encoding
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C-terminally myc-tagged PP5 was amplified using primer312 and primer313
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from pSP-mycPP5, digested with PstI and XbaI, and cloned into the
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pSP64-poly(A) vector.
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To obtain pSP-FKBP62, a DNA fragment encoding FKBP62 was
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amplified using primer306 and primer232 from pSP-mycFKBP62, digested with
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PstI and BamHI, and cloned into the pSP64-poly(A) vector. To obtain
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pSP-FKBP65, a DNA fragment encoding FKBP65 was amplified using
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primer308 and primer262 from pSP-mycFKBP65, digested with SalI and BamHI,
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and cloned into the pSP64-poly(A) vector. To obtain pSP-CYP40, a DNA
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fragment encoding CYP40 was amplified using primer310 and primer252 from
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pSP-mycCYP40, digested with PstI and BamHI, and cloned into the
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pSP64-poly(A) vector. To obtain pSP-PP5, a DNA fragment encoding PP5 was
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amplified using primer312 and primer248 from pSP-mycPP5, digested with PstI
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and XbaI, and cloned into the pSP64-poly(A) vector. To obtain pSP-TPR1, a
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DNA fragment encoding TPR1 was amplified using primer325 and primer278
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from pSP-mycTPR1, digested with PstI and BamHI, and cloned into the
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pSP64-poly(A) vector. To obtain pSP-TPR2, a DNA fragment encoding TPR2
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was amplified using primer326 and primer287 from pSP-mycTPR2, digested
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with PstI and XbaI, and cloned into the pSP64-poly(A) vector. To obtain
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pSP-TPR7, a DNA fragment encoding TPR7 was amplified using primer327 and
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primer295 from pSP-mycTPR7, digested with PstI and BamHI, and cloned into
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the pSP64-poly(A) vector.
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The plasmid pSP-mycCYP40R63A was constructed by the overlap PCR
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method using primer298, primer299, and pSP-mycCYP40 as template. The
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plasmid pSP-mycCYP40K216A was constructed by the overlap PCR method using
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primer304,
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pSP-mycCYP401-215, a DNA fragment encoding mycCYP401-215 was amplified by
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PCR using primer250 and primer301, digested with PstI and BamHI, and cloned
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into the pSP64-poly(A) vector. To obtain pSP-mycCYP40166-361, a DNA fragment
primer305,
and
pSP-mycCYP40
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as
template.
To
obtain
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encoding mycCYP40166-361 was amplified by PCR using primer300 and
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primer252, digested with PstI and BamHI, and cloned into the pSP64-poly(A)
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vector. To obtain pSP-CYP40R63A and pSP-CYP40K216A, DNA fragments
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encoding
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pSP-mycCYP40R63A and pSP-mycCYP40K216A, respectively, using primer310
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and primer252. To obtain pSP-CYP401-215, a DNA fragment encoding CYP401-215
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was amplified by PCR using primer310, primer301, and pSP-mycCYP401-215 as
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a template. To construct pSP-CYP40166-361, a DNA fragment encoding
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CYP40166-361 was amplified by PCR using primer356, primer252, and
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pSP-mycCYP40166-361 as a template.
CYP40R63A
and
CYP40K216A
were
amplified
by
PCR
from
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To obtain pSP-NtFKBP62/65, a DNA fragment encoding NtFKBP62/65
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(GeneBank accession number AB671736) was amplified by PCR using
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primer387, primer242, and template cDNA that was synthesized from N.
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tabacum (BY-2) total RNA using SuperScript III Reverse Transcriptase
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(Invitrogen, US) and primer241, digested by PstI and BamHI, and cloned into the
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pSP64-poly(A) vector. To obtain pSP-NtCYP40, a DNA fragment encoding
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NtCYP40 (GeneBank accession number AB671737) was amplified by PCR
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using primer384, primer386, and template cDNA that was synthesized from N.
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tabacum (BY-2) total RNA using SuperScript III Reverse Transcriptase
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(Invitrogen, US) and primer385, digested by SalI and BamHI, and cloned into the
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pSP64-poly(A) vector. To obtain pSP-NtPP5, a DNA fragment encoding NtPP5
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(GeneBank accession number AB671738) was amplified by PCR using
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primer381, primer383, and template cDNA that was synthesized from N.
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tabacum (BY-2) total RNA using SuperScript III Reverse Transcriptase
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(Invitrogen, US) and primer382, digested by PstI and BamHI, and cloned into the
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pSP64-poly(A) vector.
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All of the mRNAs were prepared from plasmids linearized with either
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EcoRI (FKBP65, CYP40, TPR1, TPR7, NtCYP40, NtPP5) or SmaI (FKBP62,
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PP5, TPR2, NtFKBP62/65, which have internal EcoRI sites) using the AmpliCap
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SP6 High Yield Message Maker Kit (EPICENTRE, US).
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Preparation of small RNA duplexes
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Small RNA duplexes were prepared as described previously (Iki et al, 2010),
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except that the 5'-terminal nucleotides of the guide strand siRNA of gf698-21,
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gf698-22, and miR168 (0.5 M) were phosphorylated with T4 polynucleotide
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kinase (TAKARA, Japan) by incubating with 5 M [-32P]ATP (6.9 TBq/mmol) at
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37°C for 90 min, followed by inactivation of the enzyme at 65°C for 15 min.
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Chemicals
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The stock solutions of cyclosporin A (CsA, WAKO, Japan), FK506 (SIGMA, US),
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and geldanamycin (GA, WAKO, Japan) were prepared by dissolving these
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chemicals in DMSO at a concentration of 1 mM. For use, the stock solutions
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were diluted with D.W. to 200 M, and the 200 M solutions were added to BYL
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reaction mixtures at a concentration of 20 M.
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Supplementary Figure S1
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Synthesis of N-terminally myc-tagged TPR proteins. The mRNAs encoding
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myc-TPR proteins were translated in BYL at 25°C for 90 min. The reaction
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mixtures were analyzed by immunoblotting using anti-myc antibodies.
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Supplementary Figure S2
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(A) Synthesis of non-tagged TPR domain-containing proteins. The mRNAs
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encoding TPR proteins were translated in BYL at 25°C for 90 min. The reaction
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was carried out in the presence of L-[35S]-methionine (Perkin Elmer) without
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additional amino acids. (B) Effect of the addition of CYP40 and PP5 that were
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synthesized in BYL on the production of ss gf698-21 and gf698-22 guide strands
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and miR168. AGO1 mRNA-translated BYL was mixed with mock-translated BYL
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(filled
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mRNA-translated BYL (open triangles). The mixtures were incubated with 5 nM
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small RNA duplex containing 32P-labeled siRNA guide or miRNA strands at 25°C.
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RNA was extracted at the indicated time (min), and analyzed by native PAGE.
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The concentration of produced ss small RNAs was calculated from the intensity
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of bands corresponding to ss small RNAs and duplexes. We obtained consistent
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results through three independent experiments and a typical set of results is
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shown here. (C) Synthesis of NtFKBP62/65, NtCYP40, and NtPP5 proteins. The
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mRNAs were translated in BYL at 25°C for 90 min. The reaction was carried out
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in the presence of L-[35S]-methionine (Perkin Elmer) without additional amino
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acids. (D) Effect of the addition of NtFKBP62/65, NtCYP40, and NtPP5, on the
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generation of ss siRNA. AGO1 and the N. tabacum proteins were synthesized in
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BYL, mixed, and incubated with gf698-22 siRNA duplex containing the
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32P-labeled
circles),
CYP40
mRNA-translated
BYL (open
circles),
or
PP5
guide strand at 25°C for 30 min. RNA was extracted from the
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reaction mixtures and analyzed by 15% native PAGE (left panel). Negative
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control reactions using mock-translated BYL were performed in parallel. Relative
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ss siRNA generation activity was calculated as described in the legend to Figure
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3A. The graph shows the averages and standard deviation (STD) of the relative
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ss siRNA generation activity values obtained in three independent experiments
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(right panel). Different letters indicate statistically significant differences
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(Student’s t-test, P < 0.01). (E) Effect of the addition of NtFKBP62/65, NtCYP40,
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and NtPP5, on target cleavage activity. AGO1 and the N. tabacum proteins were
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synthesized in BYL, mixed, incubated with gf698-22 siRNA duplex at 25°C for 30
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min, and further incubated with 32P-labeled GF-s target RNA. RNA was extracted
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and analyzed by denaturing 5% PAGE (left panel). Relative target cleavage
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activity was calculated as described in the legend to Figure 3B. The graph
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shows the averages and STD of the relative target cleavage activity values
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obtained in four independent experiments (right panel). Different letters indicate
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statistically significant differences (Student’s t-test, P < 0.01).
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Supplementary Figure S3
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Association of AGO1, HSP90, and small RNA duplex with C-terminally
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myc-tagged TPR proteins. FLAG-AGO1 and the C-terminally myc-tagged TPR
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proteins were synthesized in BYL, mixed, and incubated at 25°C for 30 min with
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5 nM gf698-22 siRNA duplex containing 32P-labeled guide strand in the presence
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of additional 0.75 mM ATPS and 1 mM MgCl2. The TPR-myc proteins were
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immunopurified using the anti-myc antibody and copurified RNA was extracted
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and analyzed by 15% native PAGE. To confirm recovery of the myc-tagged
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proteins and to examine the copurification of AGO1 and HSP90, a similar
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experiment using unlabeled gf698-22 siRNA duplex was performed in parallel,
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and myc-purified proteins were analyzed by immunoblotting using the anti-myc,
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anti-HSP90, and anti-AGO1 antibodies.
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Supplementary Figure S4
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(A) Effect of CsA, FK506, and GA on ss siRNA production. AGO1
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mRNA-translated BYL was incubated at 25°C for 30 min in the presence of 2%
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DMSO alone, 2% DMSO and 20 M CsA, 2% DMSO and 20 M FK506, or 2%
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DMSO and 20 M GA, and further incubated with 5 nM gf698-22 siRNA duplex
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containing
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analyzed by 15% native PAGE. A control experiment with mock-translated BYL
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was performed in parallel. Relative ss siRNA generation activity was calculated
32P-labeled
guide strand at 25°C for 30 min. RNA was extracted and
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as described in the legend to Figure 3A (‘AGO1 + DMSO’ condition = 100%).
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The graph shows the averages and STD of the relative intensity of the ss siRNA
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band obtained in three independent experiments (right panel). (B) Effect of CsA,
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FK506, and GA on target cleavage activity. AGO1 mRNA-translated BYL was
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incubated with 5 nM gf698-22 siRNA duplex at 25°C for 30 min in the presence
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of 2% DMSO, 2% DMSO and 20 M CsA, 2% DMSO and 20 M FK506, or 2%
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DMSO and 20 M GA. The reaction mixtures were further incubated with 5 nM
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internally
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and analyzed by 5% denaturing PAGE. A control experiment with the
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mock-translated BYL was performed in parallel. Relative target cleavage activity
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was calculated as described in the legend to Figure 3B (‘AGO1 + DMSO’
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condition = 100%). The graph shows the averages and STD of the relative target
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cleavage activity values obtained in three independent experiments (right panel).
32P-labeled
GF-s target RNA at 25°C for 10 min. RNA was extracted
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Supplementary Figure S5
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(A) Synthesis of non-tagged wild-type and mutant CYP40 proteins. The mRNAs
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encoding the wild-type and mutant CYP40 proteins were translated in BYL at
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25°C for 90 min. The reaction was carried out in the presence of
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L-[35S]-methionine (Perkin Elmer) without additional amino acids. (B) Effect of
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the addition of the CYP40 mutant proteins on generation of ss small RNA. AGO1
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and myc-CYP40 mutant proteins were synthesized in BYL, mixed, and
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incubated with gf698-22 siRNA duplex containing
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25°C for 30 min. After the reaction, RNA was extracted and analyzed by 15%
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native PAGE (left panel). Relative ss siRNA generation activity was calculated as
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described in the legend to Figure 3A. The graph shows the averages and STD of
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the relative ss siRNA generation activity values obtained in three independent
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experiments (right panel). Different letters indicate statistically significant
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differences (Student’s t-test, P < 0.01). (C) Effect of the addition of the CYP40
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mutant proteins on target cleavage activity. AGO1 and myc-CYP40 mutant
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proteins were synthesized in BYL, mixed, incubated with gf698-22 siRNA duplex
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at 25°C for 30 min, and further incubated with internally
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RNA at 25°C for 10 min. RNA was extracted from the mixtures and analyzed by
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5% denaturing PAGE (left panel). Relative target cleavage activity was
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32P-labeled
guide strand at
32P-labeled
GF-s target
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calculated as described in the legend to Figure 3B. The graph shows the
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averages and STD of the relative target cleavage activity values obtained in four
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independent experiments (right panel). Different letters indicate statistically
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significant differences (Student’s t-test, P < 0.01).
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Supplementary Table S1. Oligonucleotides Used in This Study
Name
Sequence (5'-3')*
primer230
GGATAATCTCTCACTGTTTTATAAG
primer231
ACTGACCTGCAGATGGAGCAGAAGCTTATTTCTGAGGAGGAT
CTTGATGCTAATTTCGAGATGCCTCCA
primer232
ACTGACGGATCCAGTGTCTCACTAACGCTCAGGTTG
primer241
GGAACACGAAAGATGCCCGATTC
primer242
ACTGACGGATCCTCCTAATGTCCACTATAACGACTC
primer246
ACTGACTGCAGATGGAGCAGAAGCTTATTTCTGAGGAGGATC
TTGAGACCAAGAATGAGAATTCTGATG
primer247
CTTATATCATTATGTTTCTGAACC
primer248
ACTGACTCTAGACTGGTCTTGAGTCTTTAGAATAGC
primer250
ACTGACCTGCAGATGGAGCAGAAGCTTATTTCTGAGGAGGAT
CTTGGTAGGTCAAAGTGTTTCATGGAC
primer251
TGTTAGTCTTCTTCCTCACTATCC
primer252
ACTGACGGATCCACCAAACCCTAAATGAAGCAGAGC
primer260
ACTGACGTCGACATGGAGCAGAAGCTTATTTCTGAGGAGGAT
CTTGAAGACGATTTCGACACGCAGAAC
primer261
CAACTCTGAAACAGTAACACACAC
primer262
ACTGACGGATCCGTTTTTGGAATCAATGCGACTCTC
primer276
ACTGACCTGCAGATGGAGCAGAAGCTTATTTCTGAGGAGGAT
CTTGTACTGATCGAATCAAGTGAGAGT
primer277
TGTATGCATCAAGAACAATAACAC
primer278
ACTGACGGATCCACATTTAAGTGGTAGAAGCAATAC
primer285
ACTGACCTGCAGATGGAGCAGAAGCTTATTTCTGAGGAGGAT
CTTTTTAACGGGTTAATGGATCCTGAG
primer286
AAAGAAGATAAAACGATGTTACAG
primer287
ACTGACTCTAGACCGAAGACTGGTTTTATCTTCACG
primer293
ACTGACCTGCAGATGGAGCAGAAGCTTATTTCTGAGGAGGAT
CTTGCGCTATGGATGGACGCTGGAGCG
primer294
AGCCCTAGCCATAACTCAGAACAG
primer295
ACTGACGGATCCGTAACAGTAATAGATTACGACAAC
primer298
TGATAACAGCATGAAATCGATTCCCCTTG
primer299
ATTTCATGCTGTTATCAAGGGGTTTATGA
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primer300
ACTGACCTGCAGATGGAGCAGAAGCTTATTTCTGAGGAGGAT
CTTGTGATCCATGACTGTGGAGA
primer301
ACTGACGGATCCCTAGACAAAATCAACAGTCTCCA
primer304
ATTTTGTCGCGGCTCATGGGAATGAGCAC
primer305
CCATGAGCCGCGACAAAATCAACAGTCTC
primer306
ACTGACCTGCAGATGGATGCTAATTTCGAGA
primer307
TGACGGATCCTCTTTTGATTAAAGATCCTCCTCAGAAATAAGC
TTCTGCTCTTCCTTACTTAGTTTCGC
primer308
ACTGACGTCGACATGGAAGACGATTTCGACAC
primer309
ACTGACGGATCCTCAAAGATCCTCCTCAGAAATAAGCTTCTG
CTCTGCCTTGGTGTCAATACTC
primer310
ACTGACCTGCAGATGGGTAGGTCAAAGTGT
primer311
ACTGACGGATCCCTAAAGATCCTCCTCAGAAATAAGCTTCTG
CTCTACGAACATTTTGCGGTAC
primer312
ACTGACCTGCAGATGGAGACCAAGAATGAG
primer313
ACTGACTCTAGAGCTGGTTAAAGATCCTCCTCAGAAATAAGC
TTCTGCTCGTTGAACATCCTGAGAAAG
primer325
ACTGACCTGCAGATGGTACTGATCGAATCAAGTGAGAGT
primer326
ACTGACCTGCAGATGGCGCTATGGATGGACGCTGGAGCG
primer327
ACTGACCTGCAGATGTTTAACGGGTTAATGGATCCTGAG
primer356
ACTGACCTGCAGATGGTGATCCATGACTGTGGAGA
primer387
ACTGACCTGCAGATGGAAGAGGATTTTGATATTCCACCG
primer384
ACTGACGTCGACATGGGAAGGCCACGGTGTTATTTGG
primer385
CTCCCAGAATGCTTGCAATTGTAC
primer386
ACTGACGGATCCAATTGCACGTGCCGGTTCCAAATC
primer381
ACTGACCTGCAGATGCCCACTATGGAAACTGAG
primer382
CTGCGGGCAAGTCAGGAAGTTGTC
primer383
ACTGACGGATCCCTCCTGATGTGCATGCACTTGGTC
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* The recognition sequences of the restriction enzymes used for cloning were
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underlined.
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Supplemental references
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Iki T, Yoshikawa M, Nishikiori M, Jaudal MC, Matsumoto-Yokoyama E, Mitsuhara
I, Meshi T, Ishikawa M (2010) In vitro assembly of plant RNA-induced silencing
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complexes facilitated by molecular chaperone HSP90. Mol Cell 39(2): 282-291
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