1. Autoantigen La Promotes Efficient RNAi, Antiviral Response, and Transposon Silencing by Facilitating Multiple-Turnover RISC Catalysis 
Ying Liu, Huiling Tan, Hui Tian, Chunyang Liang, She Chen, Qinghua Liu 
Molecular Cell 
Volume 44, Issue 3, Pages (November 2011)
DOI: /j.molcel
Copyright © 2011 Elsevier Inc. Terms and Conditions

2. Figure 1 Purification of La as an Activator of the RNAi Effector Step
(A) The ss-siRNA-initiated RISC assays were performed with HeLa extract, recombinant hAgo2, or a mixture of both as described (Ye et al., 2011).
(B) Purification of La from HeLa extract through a four-step chromatographic procedure. At the final step, individual fractions were assayed with recombinant hAgo2 and ss-siRNA for the RISC-enhancing activity (top) or resolved by SDS-polyacrylamide gel (PAGE) followed by silver-staining (bottom).
(C) Coomassie-stained SDS-PAGE showing recombinant Drosophila and human La proteins.
(D) Human minimal RISC was preassembled with recombinant hAgo2 and ss-let-7 siRNA at 37°C for 5 min and then incubated with buffer (lanes 1–3) or recombinant hLa (lanes 4–6) to reconstitute the RISC-enhancing activity.
(E and F) According to the experimental procedure in (E), duplex siRNA-initiated RISC assays were conducted using recombinant Dcr-2-R2D2 and dAgo2 alone (lanes 1 and 4) or with dLa added before (E, lanes 3 and 6) or after (L, lanes 2 and 5) RISC assembly.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
La Promotes Multiple-Turnover RISC Catalysis by Facilitating the Release of Cleaved mRNA
(A) The ss-siRNA-initiated RISC assays were performed using recombinant hAgo2 with or without hLa in a single-turnover condition (estimated concentration of RISC was ∼4 nM and target mRNA was 0.2 nM).
(B) The ss-siRNA-initiated RISC assays were performed using recombinant hAgo2 with or without hLa in a multiple-turnover condition (estimated concentration of RISC was ∼4 nM and target mRNA was 60 nM).
(C) Sequence alignment of ss-siRNA carrying 0 or 4 nt 3′ mismatches with target mRNA.
(D) A graph comparing the ratio of steady-state velocities of hAgo2-RISC in the absence or presence of hLa for two ss-siRNAs described in (C). p value was calculated by paired t test. Error bars represent standard deviations.
(E) Radiolabeled ss-siRNA/mRNA duplex was incubated with buffer, recombinant TRBP, recombinant (Rec-)hLa, or highly purified endogenous (Endo-)hLa at 37°C for 15 min.
(F) After incubating immobilized RISC/target mRNA complex with buffer or recombinant TRBP or hLa for 10 min, radiolabeled mRNA was recovered from supernatant (S) and beads (P) and resolved by 6% Urea-PAGE followed by autoradiography.
(G) The minimal hAgo2-RISC activity was assayed without or with highly purified endogenous hLa (fractions 7 and 8 in Figure 1B) in the absence (lanes 1–3) or presence (lanes 4–6) of ATP.
(H) Western blot showing that recombinant His-Hsp90 (lanes 2 and 4), but not His-hLa (lanes 1 and 3), were pulled down by ATP-Sepharose beads.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 La Associates with Ago2 and Promotes Efficient RNAi In Vivo
(A and B) Following transfection of 293T cells with Myc-hAgo2 and/or FLAG-hLa constructs, coIP experiments were performed with anti-FLAG (A) or anti-Myc (B) antibodies followed by western blotting with the corresponding antibodies.
(C) Cell extracts were treated with or without RNase before coIP experiments were performed as described above.
(D) CoIP experiments were performed using purified His-FLAG-hAgo2 and His-hLa recombinant proteins after a 30 min preincubation in the absence or presence of RNase.
(E) After siRNA-mediated knockdown in HeLa cells for 3 days, a control or RL-siRNA were cotransfected with the psiCheck2 reporter followed by dual luciferase assays in 24 hr. The efficiency of RNAi silencing was measured by the relative RL/FL luciferase activity. The graph illustrates the fold of derepression of RL-luciferase when La was depleted in HeLa, U2OS, and 293T cells. Error bars represent standard deviations.
(F) After various siRNA treatments of Teton/shMc-l U2OS cells for 2 days, Doxycycline was added to media for 2 days to induce the expression of shRNA to silence the endogenous Mcl-1 gene. The levels of Mcl-1 and α-Tubulin proteins were measured by western blotting.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 La Promotes Antiviral Response and Transposon Silencing
(A) After dsRNA-mediated depletion of Ago2, La, or both for 3 days, S2R(+) cells were transfected with a self-replicating flock house virus (FHV) mutant (FR1-ΔB2) construct (Li et al., 2004). Total RNAs were extracted 2 days after Cu2+ induction and northern blotting was performed with a probe specific to viral RNA or RP49.
(B) A graph illustrating the quantification of FHV RNA levels normalized to RP49 transcript. Error bars indicate standard deviation, n = 3.
(C) After dsRNA treatments of S2 cells for 2 days, the hp-4068 or hp construct was cotransfected with a luciferase reporter carrying the target sites for a hp-CG18854-derived endo-siRNA, and luciferase activities were monitored after 24 hr. Error bars indicate standard deviation.
(D) A graph showing the relative fold of changes in the level of ZAM transcript in S2R(+) cells following various dsRNA treatments. Different colors represent data in three independent experiments. The level of ZAM RNA was measured by real-time qPCR and normalized to RP49 transcript, n = 3. Error bars represent ± SD.
(E) The transcript levels of six different transposons were measured in various dsRNA-treated S2R(+) cells as described in (D). Error bars represent ± SD.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2011 Elsevier Inc. Terms and Conditions