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. Author manuscript; available in PMC: 2012 Nov 4.
Published in final edited form as: Mol Cell. 2011 Nov 4;44(3):502–508. doi: 10.1016/j.molcel.2011.09.011

Autoantigen La promotes efficient RNAi, antiviral response, and transposon silencing by facilitating multiple-turnover RISC catalysis

Ying Liu 1, Huiling Tan 1, Hui Tian 1, Chunyang Liang 1, She Chen 2, Qinghua Liu 1,*
PMCID: PMC3229097  NIHMSID: NIHMS333429  PMID: 22055194

SUMMARY

The effector of RNA interference (RNAi) is the RNA-induced silencing complex (RISC). C3PO promotes the activation of RISC by degrading Argonaute2 (Ago2)-nicked passenger strand of duplex siRNA. Active RISC is a multiple-turnover enzyme that uses the guide strand of siRNA to direct Ago2-mediated sequence-specific cleavage of complementary mRNA. How this effector step of RNAi is regulated is currently unknown. Here, we used human Ago2 minimal RISC system to purify Sjögren’s syndrome antigen B (SSB)/autoantigen La as an activator of the RISC-mediated mRNA cleavage activity. Our reconstitution studies showed that La could promote multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from RISC. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC.

Keywords: RNAi, RISC, Ago2, La, multiple-turnover

INTRODUCTION

RNA interference (RNAi), in all of its manifestations, impacts many aspects of eukaryotic gene expression and is involved in numerous biological and disease processes. The canonical form of RNAi is a conserved post-transcriptional gene silencing mechanism, whereby double-stranded RNA (dsRNA) induces sequence-specific degradation of complementary mRNA (Fire et al., 1998). The ribonuclease (RNase) III Dicer complex initiates RNAi response by processing long dsRNA into ~21-nucleotide (nt) siRNA duplexes (Bernstein et al., 2001; Elbashir et al., 2001; Liu et al., 2003). In plants and animals, the exogenous siRNAs function as a potent defense mechanism against invading nucleic acids, such as viruses and transgenes (Ding and Voinnet, 2007). Additionally, endogenous (endo)-siRNAs can be derived from transposable elements, complementary annealed transcripts, and long fold-back transcripts within the genome (Okamura and Lai, 2008). These endo-siRNAs play important roles in diverse biological processes, including transposon silencing in Drosophila somatic cells (Okamura and Lai, 2008).

Activation of the effector RNA-induced silencing complex (RISC) involves a three-step process in Drosophila and mammals (Liu and Paroo 2010). First, nascent siRNA duplex (guide strand-passenger strand) is recruited to the catalytic subunit of RISC, Argonatute2 (Ago2), by Dicer-2(Dcr-2)-R2D2 complex (Liu et al., 2003; Pham et al., 2004; Tomari et al., 2004) and/or heat shock proteins (Iwasaki et al. 2010; Miyoshi et al. 2010). Second, Ago2, an endonuclease, nicks the passenger strand into 9-nt and 12-nt fragments (Matranga et al., 2005; Miyoshi et al., 2005; Rand et al., 2005). Finally, C3PO, another endonuclease, activates RISC by degrading Ago2-nicked passenger fragments (Liu et al., 2009; Ye et al. 2011).

Active RISC is a multiple-turnover enzyme that uses single-stranded (ss)-siRNA to guide Ago2-mediated sequence-specific cleavage of complementary mRNA (Haley and Zamore, 2004; Liu et al., 2004; Martinez et al., 2002; Rivas et al., 2005). How this effector step of RNAi is regulated is currently unknown. A recent study suggests that the release of cleaved mRNA from RISC is a rate-limiting step for multiple-turnover RISC catalysis (Haley and Zamore, 2004). In this study, we identified autoantigen La as a RNAi activator that could promote the multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA from Ago2. Moreover, we demonstrated that La was required for efficient RNAi, antiviral defense, and transposon silencing in vivo. Taken together, the findings of C3PO and La reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC.

RESULTS

Purification of La as an activator of the RNAi effector step

To study regulation of the RNAi effector step, we used recombinant human Ago2 (hAgo2) and ss-siRNA to assemble minimal RISC in vitro and search for new regulators of RISC activity by supplementing mammalian cell extract. Addition of cytoplasmic (S100) extract of HeLa cells greatly enhanced the RISC-mediated mRNA cleavage activity (Figure 1A), suggesting the presence of an activator of RISC. This RISC activator was subsequently purified to homogeneity through a four-step chromatographic procedure (see supplementary methods). At the final step, a single ~50 kDa protein closely correlated with the RISC-enhancing activity and was identified as autoantigen La, also known as Sjogren’s syndrome antigen B (SSB), by mass spectrometric analysis (Figure 1B).

Figure 1.

Figure 1

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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 pre-assembled with recombinant hAgo2 and ss-let-7 siRNA at 37 ºC for 5 min, and then incubated with buffer (lanes 1–3) or recombinant hLa (lanes 4–6) to reconstitute the RISC-enhancing activity. (E–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.

Sjögren’s syndrome is a chronic autoimmune disorder in which immune cells attack and destroy the exocrine glands that produce tears and saliva (Srinivasan and Slomovic, 2007). A hallmark of Sjögren’s syndrome is the production of antibodies against self-antigens, such as autoantigen La (Mattioli and Reichlin, 1974). The evolutionarily conserved La protein plays important roles in fundamental biological processes from yeast to human, including RNA polymerase III (Pol III) transcription, tRNA biogenesis, mRNA stabilization, and translational regulation (Wolin and Cedervall, 2002).

We generated polyhistidine-tagged Drosophila (d) and human (h) La recombinant proteins to reconstitute the RISC-enhancing activity (Figure 1C). Consistent with the purification result, addition of recombinant hLa greatly enhanced hAgo2 minimal RISC activity (Figure 1D). Because dAgo2 exhibited little ss-siRNA-initiated RISC activity (Figure S1), we assembled Drosophila RISC by incubating recombinant Dcr-2-R2D2 and dAgo2 with duplex siRNA followed by mild heat treatment to inactivate free dAgo2 and block further RISC assembly (Figure 1E) (Liu et al., 2009). Robust RISC-enhancing activity was observed no matter when dLa was added before or after the RISC assembly (Figure 1F), suggesting that La primarily regulated the RISC activity rather than RISC assembly.

La promotes multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA

The RISC-mediated mRNA cleavage activity can potentially be regulated at three steps: target recognition, mRNA cleavage, and product release. Regulation of either target recognition or mRNA cleavage should affect the rate of both single-turnover and multiple-turnover RISC catalysis. By contrast, regulation of product release should only affect the rate of the multiple-turnover reaction. In a single-turnover condition (RISC was in excess over target mRNA), we observed an identical rate of mRNA cleavage in the absence or presence of La (Figure 2A). In a multiple-turnover condition (target mRNA was in large excess), a burst of cleavage products was detected early in the reaction (single-turnover phase), and was followed by a slower steady-state velocity of target cleavage (multiple-turnover phase) in the absence of La (Figure 2B). Addition of La specifically enhanced the efficiency of the RISC-mediated mRNA cleavage in the multiple-turnover phase (Figure 2B). These kinetic studies indicate that La promotes the multiple-turnover RISC catalysis by facilitating the release of cleaved mRNA.

Figure 2.

Figure 2

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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 ~4nM and target mRNA was 0.2nM). (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 ~4nM and target mRNA was 60nM). (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. (E) Radiolabeled ss-siRNA/mRNA duplex was incubated with buffer, recombinant TRBP, recombinant (Rec-)hLa, or highly purified endogenous (Endo-)hLa at 37 ºC for 15min. (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 (lane 1 and 3), were pulled down by ATP-Sepharose beads.

The product release from RISC can be facilitated by weakening the interaction between the 3’ end of guide strand and target mRNA (Haley and Zamore, 2004). Thus, we compared the steady-state velocities of RISC reactions in the absence and presence of La with the use of ss-let-7 siRNA carrying zero or four nucleotide 3’ mismatches with the target mRNA (Figure 2C). In support of our conclusion, introducing mismatches at the 3’ end of guide strand significantly mitigated the RISC-enhancing effect of La (Figure 2D).

We found that highly purified recombinant and endogenous human La could facilitate the dissociation of target mRNA from the guide strand in a ss-siRNA/target mRNA duplex (Figure 2E). In contrast, endogenous hLa was unable to dissociate the passenger strand from guide strand in an siRNA duplex with 2-nt 3’ overhang (Figure S2A). These findings are consistent with previous report that mammalian La could dissociate RNA-RNA hybrids with 5’ or 3’ long overhangs (Huhn et al., 1997). Moreover, we loaded immobilized hAgo2-RISC with radiolabeled target mRNA to test whether La could promote mRNA release from RISC. Indeed, incubation with recombinant hLa resulted in the rapid release of cleaved mRNA from immobilized RISC into the supernatant (Figure 2F). However, this was not the case with TRBP, a dsRNA-binding partner for Dicer in the human RNAi pathway (Chendrimada et al., 2005; Haase et al., 2005). The data were consistent with that TRBP lacked the RISC-enhancing activity (Figure S2B). Taken together, these results indicate that La facilitates the release of cleaved mRNA from RISC.

It was recently reported that ATP was required for multiple-turnover RISC catalysis in Drosophila embryo extract (Haley and Zamore, 2004). However, there was no appreciable effect of ATP on the RISC-enhancing activity of highly purified endogenous or recombinant hLa even after ATP depletion (Figure 2G and Figure S2C). Consistently, we could not observe the binding of recombinant hLa to ATP-Sepharose (Figure 2H), nor detect any known ATP-binding motif in the La protein by bioinformatics analysis (data not shown). Thus, we concluded that La promoted the multiple-turnover RISC catalysis in an ATP-independent manner. It is possible that another ATP-dependent factor may function redundantly or together with La to promote Drosophila RISC catalysis.

La associates with Ago2 and promotes efficient RNAi in vivo

We detected stable association between Myc-Ago2 and Flag-La in 293T cells by reciprocal co-immunoprecipitation (IP) experiments (Figures 3A and 3B). Treatment of cell extracts with RNase abolished this interaction, suggesting that La associates with Ago2 in a RNA-dependent manner (Figure 3C, compare lane 2 and lane 4). The interaction between human La and Ago2 was confirmed by co-IP experiments using purified recombinant proteins (Figure 3D). It was previously reported that recombinant Ago2 co-purified with contaminating RNA (MacRae et al., 2008; Wang et al., 2009). Consistently, RNase treatment of recombinant proteins also abolished the association between Ago2 and La (Figure 3D, compare lane 2 and lane 4).

Figure 3.

Figure 3

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La associates with Ago2 and promotes efficient RNAi in vivo. (A–B) Following transfection of 293T cells with Myc-hAgo2 and/or Flag-hLa constructs, co-IP 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 co-IP experiments were performed as described above. (D) Co-IP experiments were performed using purified His-Flag-hAgo2 and His-hLa recombinant proteins after a 30min pre-incubation in the absence or presence of RNase. (E) After siRNA-mediated knockdown in HeLa cells for three days, a control or RL-siRNA were co-transfected with the psiCheck2 reporter followed by dual luciferase assays in twenty-four hours. The efficiency of RNAi silencing was measured by the relative RL/FL luciferase activity. The graph illustrates the fold of de-repression 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 two days, Doxycycline were added to media for two 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.

To examine if La promoted efficient RNAi in vivo, we performed siRNA-mediated knockdown of La expression in HeLa cells (Figure S3A), followed by co-transfection of a dual [Firefly (FL) and Renilla (RL)] luciferase reporter and a control or RL-siRNA. Consistent with our biochemical studies, depletion of La or Ago2 significantly compromised the efficiency of RNAi silencing in vivo (Figure S3B). A similar phenotype was observed in HeLa, U2OS, and 293T cells, wherein the lack of La protein typically resulted in a 5 to 10-fold de-repression of RL-luciferease (Figure 3E). Moreover, we obtained a Teton-inducible U2OS cell line, in which induction of a short hairpin RNA (shMcl-1) could effectively silence the expression of endogenous Mcl-1 protein. Again, depletion of La or Dicer restored Mcl-1 expression in the presence of Doxycycline (Figure 3F). Taken together, these genetic studies indicate that La is required for efficient RNAi silencing of exogenous and endogenous genes in mammalian cells.

La promotes antiviral response and transposon silencing

RNAi serves as an important antiviral defense mechanism in plants and animals (Ding and Voinnet, 2007). To study the role of La in the antiviral response, we used dsRNA treatment to specifically knockdown the expression of Ago2, La, or both in Drosophila S2 cells (Figures S4A and S4B), and transfected these cells with a self-replicating flock house virus (FHV) mutant (FR1-ΔB2) construct (Li et al., 2004). The lack of B2, a suppressor of RNAi, allowed S2 cells to effectively repress viral expression through RNAi (Li et al., 2004). While knockdown of either Ago2 or La resulted in ~1.5-fold accumulation of FHV RNA, depletion of both proteins caused a ~2.5-fold induction of viral RNA expression (Figures 4A and 4B). Thus, La is involved in the RNAi-mediated antiviral response in Drosophila cells.

Figure 4.

Figure 4

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La promotes antiviral response and transposon silencing. (A) After dsRNA-mediated depletion of Ago2, La, or both for three 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 two 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 two days, the hp-4068 or hp-18854 construct was co-transfected with a luciferase reporter carrying the target sites for a hp-CG18854-derived endo-siRNA, and luciferase activities were monitored after twenty-four hours. 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.

The long hairpin (hp) transcripts-derived endo-siRNAs can be loaded onto Ago2 to cleave complementary target mRNA (Czech et al., 2008; Ghildiyal et al., 2008; Okamura et al., 2008). In S2 cells, ectopic expression of hp-CG18854, but not hp-CG4068, repressed the expression of a luciferase reporter carrying target sites for a hp-CG18854-derived siRNA (Figure 4C) (Okamura et al., 2008). Depletion of Ago2 or La significantly attenuated the endo-siRNA-mediated luciferase repression (Figure 4C). These results indicate that La also plays a critical role in the Drosophila endo-siRNA pathway.

An abundant source of endo-siRNAs is the transposable elements, whose activities must be silenced to protect genomic integrity and organism survival (Czech et al., 2008; Ghildiyal et al., 2008; Tam et al., 2008; Saito and Siomi 2010). In Drosophila, these endo-siRNAs contribute critically to transposon silencing in somatic cells (Czech et al., 2008; Ghildiyal et al., 2008). Thus, we knocked down the expression of La, Ago2, or both in S2 cells followed by quantitative RT-PCR to compare the expression level of retrotransposon ZAM. While knockdown of La or Ago2 caused, respectively, a ~2- or 5-fold accumulation of ZAM transcript, depletion of both proteins resulted in up to 20-fold induction of the transposon expression (Figure 4D). This synergistic effect further suggested that Ago2 and La worked cooperatively at the RNAi effector step. Moreover, we observed significant accumulation of the transcripts of Idefix, DOC, gypsy6 and 412, but not those of DM297 and BEL1, when La and/or Ago2 were depleted (Figure 4E), suggesting that La was broadly involved in the endo-siRNA-mediated transposon silencing.

Discussion

In this study, we took a biochemical fractionation and reconstitution approach to identify autoantigen La as a new activator of the RNAi effector step. The biological importance of this study is underscored by genetic findings that La is required for efficient RNAi in mammalian cells, and promotes antiviral defense, and transposon silencing in Drosophila cells. Moreover, our studies showed that La specifically promoted the multiple-turnover RISC catalysis, but not single-turnover RISC catalysis. Consistent with this function, La could associate with Ago2 in a RNA-dependent fashion, dissociate target mRNA from the guide strand, and facilitate the release of cleaved mRNA from RISC. We find it intriguing that La facilitates the unwinding of ss-siRNA/target mRNA duplex even though it shares no similarity to any known helicases. One possible explanation is that La may separate the two strands of dsRNA by trapping single-stranded RNA in a manner reminiscent of Adenovirus single-stranded DNA binding protein (DBP) that uses the force of multimerization to drive ATP-independent DNA unwinding (Dekker et al., 1997). Future investigation is warranted to clarify the exact mechanism by which La promotes strand separation of dsRNA and release of cleaved mRNA from RISC.

The interaction among Ago2, siRNA, and target mRNA within RISC is a highly dynamic process involving significant conformational changes in both the protein and RNA components (Wang et al., 2009; Parker 2010). During RISC assembly, Ago2 receives duplex siRNA, nicks the passenger strand, and then C3PO degrades the passenger fragments to activate RISC (Liu et al., 2009; Ye et al. 2011). During the RISC-mediated mRNA cleavage, target mRNA comes in to form a duplex with guide RNA, Ago2 slices target mRNA, and then La promotes the release of cleaved mRNA to initiate another round of RISC catalysis. While these are two similar processes, C3PO primarily functions in RISC assembly because it prefers to degrade small RNA rather than long mRNA (Ye et al. 2011)(data not shown). In contrast, La specifically promotes multiple-turnover of RISC catalysis because it can promote strand separation of the ss-siRNA/target mRNA duplex, but not a duplex siRNA. Taken together, these studies reveal a general concept that regulatory factors are required to remove Ago2-cleaved products to assemble or restore active RISC. Furthermore, the findings of La’s role in RNAi may provide fresh insights into many previously reported functions of La in cellular processes, and the pathogenesis of autoimmune diseases that frequently produce autoantibodies against La (Mattioli and Reichlin, 1974).

Materials and Methods

Minimal RISC assay

Recombinant human Ago2 protein was first incubated with ss-let-7 siRNA at 37 ºC for 5 min to pre-assemble minimal RISC. A 5’G-cap labeled 300 nt RNA containing a perfect let-7 target site was then added into reaction as the RISC substrate. To insure multiple-turnover condition, we used ~0.5nM RISC and 4nM target mRNA in the RISC assay. The active RISC concentration was estimated through a burst kinetics method (Haley and Zamore, 2004). After incubating at 37 ºC for the indicated time, the reactions were stopped by addition of 200 μl 0.3 M NaOAc, phenol/chloroform extracted, ethanol precipitated with glycogen as a carrier, and resolved on a denaturing 6% polyacrylamide gel.

target mRNA/ss-siRNA dissociation assay

The ss-siRNA was 5’ 32P-radiolabeled by T4 polynucleotide kinase, PAGE purified, and annealed with 1.5 fold excess of cold target mRNA or complementary siRNA. The dissociation assays (10 μl) were carried out with different proteins in the RISC buffer essentially as described (Nykanen et al., 2001). RNAs were extracted and resolved on a 16% native PAGE gel.

mRNA release assay

Recombinant His-FLAG-hAgo2 was incubated with anti-Flag M2 magnetic beads at 4 ºC for 1 hour to immobilize hAgo2. Beads were then extensively washed with Buffer A and loaded with ss-siRNA and radiolabeled target mRNA at 37 ºC for 20 min to assemble RISC and initiate mRNA cleavage reaction. After extensive wash to remove unbound target mRNA, immobilized RISC/target mRNA was then incubated with buffer alone or different recombinant proteins at 37 ºC for 10 min to assay for the release of cleaved mRNA into the supernatant. RNAs from supernatant (S) and beads (P) were extracted and resolved on 6% denaturing PAGE gel.

In vivo RNAi assay

In 24-well, approximately 2.5×104 HeLa cells were transfected with different siRNAs by lipofectamine RNAiMax (Invitrogen). After three days, cells were again co-transfected with psiCHECK2 (Promega) and GFP- or RL-siRNA by lipofectamine2000. Twenty-four hours after transfection, cells were lysed and subjected to dual luciferase assay (Promega). Every experiment has triplicate transfections for each sample. For the Teton-inducible U2OS/shMcl-1 cells, 4×105 cells were transfected with different siRNAs by RNAiMax in 6-well plate. Three days later, the same siRNA transfection was repeated and Doxycycline was added to the media to induce the expression of Mcl-1shRNAs. Twenty-four hours after induction, cells were harvested to prepare protein extracts and perform Western blotting to compare the level of Mcl-1 protein.

Antiviral response in S2 cells

The antiviral response was measured in S2 cells essentially as described (Li et al., 2004). In 12-well, 1×106 S2R(+) cells cultured in serum-free SFX medium (Hyclone) were transfected with 300ng pFR1-ΔB2 plus 300ng different dsRNAs (eg. dsFluc, dsAgo2 and dsLa) using cellfectin reagent (Invitrogen). Two days after viral RNA induction, cells were harvested and total RNAs were extracted by Trizol reagent (Invitrogen). 6μg RNA samples were then analyzed by Northern blot (NorthernMax kit, Ambion). DNA fragments corresponding to the FHV or RP49 were labeled as probes using Rediprime II random prime labeling system (Amersham BioSciences).

Supplementary Material

01

HIGHLIGHTS.

  1. Autoantigen La is a novel activator of the RISC-mediated mRNA cleavage activity.

  2. La promotes the multiple-turnover, but not single-turnover, RISC catalysis.

  3. La promotes efficient RNAi, antiviral defense, and transposon silencing in vivo.

Acknowledgments

We thank Drs. X. Wang, S. W. Ding and E. C. Lai for useful reagents, Drs. Y. Liu and N. Conrad for discussion and reading the manuscript, and Y. Duan and Y. Zhang for technical assistance. Y. L. is supported by the Sara and Frank McKnight student fellowship. The work is supported by a Welch grant (I-1608) and the National Institute of Health grants awarded to Q. L. (GM078163 and GM084010).

Footnotes

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