The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing
2013; Springer Nature; Volume: 32; Issue: 8 Linguagem: Inglês
10.1038/emboj.2013.52
ISSN1460-2075
AutoresLukas Stalder, Wolf Heusermann, Lena Sokol, Dominic Trojer, Joël Wirz, Justin Hean, Anja Fritzsche, Florian Aeschimann, Vera Pfanzagl, Pascal Basselet, Jan Weiler, Martin Hintersteiner, Dylan Morrissey, Nicole Meisner‐Kober,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle19 March 2013Open Access Source Data The rough endoplasmatic reticulum is a central nucleation site of siRNA-mediated RNA silencing Lukas Stalder Corresponding Author Lukas Stalder Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, SwitzerlandPresent address: Swiss Group for Clinical Cancer Research, Bern, Switzerland. Search for more papers by this author Wolf Heusermann Wolf Heusermann Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Lena Sokol Lena Sokol Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Dominic Trojer Dominic Trojer Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Joel Wirz Joel Wirz Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Justin Hean Justin Hean Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, SwitzerlandPresent address: University of the Witwatersrand Medical School, School of Pathology, Johannesburg, South Africa. Search for more papers by this author Anja Fritzsche Anja Fritzsche Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Florian Aeschimann Florian Aeschimann Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Friedrich Miescher Institute, Basel, Switzerland Search for more papers by this author Vera Pfanzagl Vera Pfanzagl Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Pascal Basselet Pascal Basselet Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Jan Weiler Jan Weiler Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Martin Hintersteiner Martin Hintersteiner Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author David V Morrissey David V Morrissey Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Cambridge, MA, USA Search for more papers by this author Nicole C Meisner-Kober Corresponding Author Nicole C Meisner-Kober Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Lukas Stalder Corresponding Author Lukas Stalder Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, SwitzerlandPresent address: Swiss Group for Clinical Cancer Research, Bern, Switzerland. Search for more papers by this author Wolf Heusermann Wolf Heusermann Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Lena Sokol Lena Sokol Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Dominic Trojer Dominic Trojer Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Joel Wirz Joel Wirz Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Justin Hean Justin Hean Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, SwitzerlandPresent address: University of the Witwatersrand Medical School, School of Pathology, Johannesburg, South Africa. Search for more papers by this author Anja Fritzsche Anja Fritzsche Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Florian Aeschimann Florian Aeschimann Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Friedrich Miescher Institute, Basel, Switzerland Search for more papers by this author Vera Pfanzagl Vera Pfanzagl Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Pascal Basselet Pascal Basselet Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Jan Weiler Jan Weiler Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Martin Hintersteiner Martin Hintersteiner Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author David V Morrissey David V Morrissey Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Cambridge, MA, USA Search for more papers by this author Nicole C Meisner-Kober Corresponding Author Nicole C Meisner-Kober Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland Search for more papers by this author Author Information Lukas Stalder 1, Wolf Heusermann1, Lena Sokol1, Dominic Trojer1, Joel Wirz1, Justin Hean1, Anja Fritzsche1, Florian Aeschimann1,2, Vera Pfanzagl1, Pascal Basselet1, Jan Weiler1, Martin Hintersteiner1, David V Morrissey3 and Nicole C Meisner-Kober 1 1Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Basel, Switzerland 2Friedrich Miescher Institute, Basel, Switzerland 3Novartis Institutes for Biomedical Research, NIBR Biologics Center, RNAi Therapeutics, Cambridge, MA, USA *Corresponding authors. Novartis Institutes for Biomedical Research, Developmental and Molecular Pathways, Novartis Campus, Fabrikstrasse 22.2.023, Basel 4000, Switzerland. Tel.:+41 76 3981721; E-mail: [email protected] or Tel.:+41 79 4099798; Fax:+41 61 6967470; E-mail: [email protected] The EMBO Journal (2013)32:1115-1127https://doi.org/10.1038/emboj.2013.52 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Despite progress in mechanistic understanding of the RNA interference (RNAi) pathways, the subcellular sites of RNA silencing remain under debate. Here we show that loading of lipid-transfected siRNAs and endogenous microRNAs (miRNA) into RISC (RNA-induced silencing complexes), encounter of the target mRNA, and Ago2-mediated mRNA slicing in mammalian cells are nucleated at the rough endoplasmic reticulum (rER). Although the major RNAi pathway proteins are found in most subcellular compartments, the miRNA- and siRNA-loaded Ago2 populations co-sediment almost exclusively with the rER membranes, together with the RISC loading complex (RLC) factors Dicer, TAR RNA binding protein (TRBP) and protein activator of the interferon-induced protein kinase (PACT). Fractionation and membrane co-immune precipitations further confirm that siRNA-loaded Ago2 physically associates with the cytosolic side of the rER membrane. Additionally, RLC-associated double-stranded siRNA, diagnostic of RISC loading, and RISC-mediated mRNA cleavage products exclusively co-sediment with rER. Finally, we identify TRBP and PACT as key factors anchoring RISC to ER membranes in an RNA-independent manner. Together, our findings demonstrate that the outer rER membrane is a central nucleation site of siRNA-mediated RNA silencing. Introduction Since the discovery of RNA interference (RNAi), the interest in small RNAs as both therapeutic targets and agents has been growing rapidly. The effective and safe delivery of small RNA therapeutics into cells remains one of the biggest challenges, which is partially linked to the still incomplete picture of the intracellular sites of endogenous RNA silencing. Small-interfering RNAs (siRNAs) and microRNAs (miRNAs) exhibit their functions once they are loaded into RNA induced silencing complexes (RISCs) (Carthew and Sontheimer, 2009). Proteins from the Argonaute (Ago) family are the core of RISCs, and Argonaute2 (Ago2) is the only of the four mammalian Ago proteins with the ability to slice the target mRNA by endonucleolytic cleavage (Liu et al, 2004; Meister et al, 2004). Although Ago2 can bind single-stranded siRNAs in vitro (Rivas et al, 2005), endogenous loading of double-stranded small RNAs is thought to require the RISC loading machinery (Liu et al, 2004; Yoda et al, 2010). The canonical, minimal human RISC loading complex (RLC) comprises Ago2, Dicer and TAR RNA binding protein (TRBP) (Gregory et al, 2005; Maniataki and Mourelatos, 2005; MacRae et al, 2008; Noland et al, 2011). This triad of proteins is capable of binding and processing dsRNA into 21–23nt siRNAs or miRNAs, loading of Ago2 and removing the passenger strand (MacRae et al, 2008). As Dicer knockout mouse embryonic stem (ES) cells—while devoid of mature miRNAs—are however proficient of siRNA-mediated gene silencing (Kanellopoulou et al, 2005), it has been suggested that this canoncial mode of RISC loading can be bypassed by other mechanisms, one of them involving the Heat shock cognate 70 (Hsc70) and Heat shock protein 90 (Hsp90) chaperones (Miyoshi et al, 2005; Iki et al, 2010; Iwasaki et al, 2010; Johnston et al, 2010; Miyoshi et al, 2010). Once Ago2 is loaded with the double-stranded siRNA, only one strand (guide) is retained and the other strand (passenger) gets removed and degraded ( Matranga et al, 2005; Rand et al, 2005; Leuschner et al, 2006; Miyoshi et al, 2010), which can be facilitated by a complex consisting of TRAX and translin (C3PO, component 3 promoter of RISC) (Liu et al, 2009; Ye et al, 2011). The specific subcellular sites of the RISC loading, target association and mRNA silencing steps remain under debate. Ago2, miRNAs and target mRNAs that are targeted for translational inhibition have been found to localize to P-bodies (Liu et al, 2005; Pillai et al, 2005; Jagannath and Wood, 2009), and it has been suggested that miRNAs and RNAi proteins guide their target mRNAs to P-bodies (Jakymiw et al, 2005; Pillai et al, 2005; Eulalio et al, 2007b). However, microscopically visible P-bodies do not seem to be required for RNAi (Chu and Rana, 2006; Eulalio et al, 2007b), but have been proposed to be rather a consequence than a cause of silencing (Eulalio et al, 2007a, 2007b). Moreover, siRNAs have been found to localize to P-bodies as double strands in an at least partially Ago2-dependent manner (Jakymiw et al, 2005; Jagannath and Wood, 2009). Other reports have demonstrated a link between RNAi and membranes (Cikaluk et al, 1999; Tahbaz et al, 2001, 2004; Gibbings et al, 2009; Lee et al, 2009; Gibbings and Voinnet, 2010). In early reports, Dicer and Ago2 have been shown to fractionate with membranes (Tahbaz et al, 2004) and to co-localize with the Golgi apparatus (Cikaluk et al, 1999; Tahbaz et al, 2001; Barbato et al, 2007). Furthermore, disruption of the Hermansky Pudlak 1 and 4 proteins (HPS1, HPS4), which are implicated in membrane trafficking and function (Huizing et al, 2000), accelerate the loading of Ago2 with siRNAs in flies (Lee et al, 2009). Additionally, it has been proposed that RISC assembly and disassembly is linked to membranes of the endo-lysosomal system (Gibbings et al, 2009; Lee et al, 2009; Gibbings and Voinnet, 2010). Given that there is still no clear picture about the sites of RISC loading, target mRNA association and silencing, in this work we aimed to quantitatively and spatially follow the siRNA fate within the cell upon lipid delivery from initial uptake and subcellular redistribution to its entry into the RNAi pathway, and to identify the sites of RNAi activity. Results Ago2, siRNAs and miRNAs localize to a number of different compartments To characterize the intracellular distribution of RNAi pathway proteins, exogenously added siRNAs, and endogenous miRNAs, HeLa cells were transfected by lipofection with siRNAs against SSB (Pei et al, 2010) (Sjogren syndrome antigen B), lysed after 24 h and the post-nuclear detergent-free supernatants were fractionated on continuous density sucrose gradients (Figure 1A). Markers for lysosomes (Lamp2), endosomes and multi-vesicular bodies (MVBs; Hrs, Rab5, Tsg101) were enriched in fractions 2 and 3 (20–33% sucrose), markers for Golgi (β4-GalT1) in fractions 3–5, the ER-Golgi intermediate compartment (ERGIC; p58), the ER (Calnexin) and ribosomes (RPS6) in fractions 4–7 (42–64% sucrose), confirming the expected fractionation pattern consistent with fractionation of cytoplasmic lysates in previous studies (e.g., Gibbings et al, 2009; Jouannet et al, 2012). Ago2 showed a broad distribution and was present throughout fractions 3–9. Similarly, Ago1, Dicer and PACT fractionated broadly, whereas only TRBP showed evidence for a more confined localization and co-sedimented sharply with Golgi and ER marker proteins (Figure 1A). The fractionation of the RNAi pathway proteins did not change upon the transfection of an siRNA, suggesting that a transfected siRNA does not induce a major redistribution of these proteins (Supplementary Figure S1A). Figure 1.Ago2 and siRNAs localize to a number of different compartments. (A) Western blot analysis of sucrose gradient fractions. (B) Immune fluorescence of HeLa cells stained with anti-Ago2 and counterstained with antibodies against marker proteins for P-bodies (Dcp1), ER (Calreticulin), Golgi (β4-GalT1), ERGIC (p58) and early Endosomes (Hrs). Settings were chosen so that no background was detected from cells stained only with secondary antibodies, all images were taken with the same settings. (C) RT–qPCR quantification of total intracellular SSB guide strand (left panel) and in the sucrose fractions (right panel) at the indicated time points post transfection. (D) Life cell imaging of HeLa cells transfected with 5′TMR-labelled HuR siRNAs (green) and counterstained with Lysotracker (lysosome marker; blue) or transferrin (endosome marker; red). Source Data for Figure 1 [embj201352-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint To confirm the general fractionation pattern of endogenous Ago2, we performed immune fluorescence (IF) of Ago2 as well as the different organelle marker proteins in HeLa cells (Figure 1B). The established high specificity of the Ago2 antibody (clone 11A9) (Rüdel et al, 2008) was validated also in our hands using a peptide comprising the antigenic epitope (Supplementary Figure S1B). Ago2 staining showed the typical diffuse punctuate pattern throughout the cytoplasm with the typical enrichment in the perinuclear region and a half-moon shaped structure (Golgi) and some individual strong foci (P-bodies). In accordance with the broad fractionation of Ago2, endogenous Ago2 also co-localized by IF partially with the ER, ERGIC, Golgi, P-bodies and early endosomes (Figure 1B). This intracellular distribution of endogenous Ago2 is consistent with previous publications, where Ago2 was shown to partially co-localize with Golgi (Cikaluk et al, 1999; Tahbaz et al, 2001), P-bodies (Liu et al, 2005; Pillai et al, 2005; Sen and Blau, 2005; Leung et al, 2006; Ohrt et al, 2008; Zeng et al, 2008; Jagannath and Wood, 2009; Pare et al, 2009) or endosomes (Gibbings et al, 2009; Lee et al, 2009). Additionally, our data confirm the general notion that endogenous Ago2 appears to distribute differently than Ago2-GFP, which was shown to strongly accumulate in P-bodies (Liu et al, 2005; Sen and Blau, 2005; Leung et al, 2006; Ohrt et al, 2008; Jagannath and Wood, 2009). Consistent with an accumulation in P-bodies, we found overexpressed Ago2-GFP enriched in the Dcp1 containing fractions (4–6) of the continuous sucrose density gradient (Supplementary Figure S1C). As the overall Ago2 localization was not instructive about sites of RNA silencing activity, we next followed the siRNA during onset of uptake and a potential subsequent subcellular redistribution to monitor its putative entry into the RNAi pathway. HeLa cells were transfected with the SSB siRNA and a control siRNA with no target in human cells (pGL3), harvested after 1.3, 3.3, 5 and 28 h and fractionated as above. During the 5-h transfection period, the SSB siRNA was increasingly enriched in the endo-/lysosomal fractions. After 28 h, the cells were cleared of 75–90% of the transfected siRNAs by secretion and/or degradation (Figure 1C; Supplementary Figure S1D), whereas the remaining siRNA was now mostly in the non-endosomal fractions, suggesting that only 10–25% of the transfected siRNA ever had the potential to load into RISC. In accordance with a strong accumulation and degradation of the siRNA in endosomes and lysosomes, respectively, a transfected TMR-labelled siRNA co-localized with endosomal (transferrin) and lysosomal (LysoTracker) markers in living cells and co-sedimented with these organelles in sucrose gradients (Figure 1D; Supplementary Figure S1E–G), suggesting that the bulk of transfected siRNA enters the cells through the endosomal system (Lu et al, 2009), and that the major fraction of the transfected siRNA is quickly targeted to lysosomes for degradation. Interestingly, the non-targeting pGL3 siRNA fractionated similar to the SSB siRNA, suggesting that bulk trafficking of siRNAs is not driven by the presence of a target mRNA (Supplementary Figure S1D). Given this dramatic clearance of siRNA within the initial phase of transfection, we reasoned that at late time points, the remaining amount of siRNA might better reflect the active population. Indeed, siRNA remaining at 28 h fractionated in a sucrose gradient similarly as endogenous miR-16 (Supplementary Figure S1H), a representative and relatively abundant miRNA in HeLa cells, indicating that, once in the cytoplasm, siRNA is subject to similar protein interactions and trafficking events as endogenous miRNAs. However, both, miR-16 and the SSB siRNA were present in all Ago2 containing fractions, suggesting that even after initial clearance, the co-fractionation of RNAi factors and siRNAs or miRNAs is also not indicative to discern the active siRNA/Ago2 population. Excess of inactive siRNA masks active population Using Ago2 immune precipitations (IPs), we next quantified how much of the siRNA residing in the cell after the initial major clearance wave is loaded into Ago2. Ago2 IPs (Rüdel et al, 2008) were quantitatively highly reproducible (Supplementary Figure S2A and B), and importantly, showed a linear correlation between eluted Ago2 and the amount of input lysate over the relevant range of sample concentration (Supplementary Figure S2C). Also, the IP efficiency of Ago2 was independent of the sucrose concentration as confirmed for both, the continuous (Supplementary Figure S2D) and discontinuous sucrose gradients (Supplementary Figure S2E) used in this study. Additionally, to rule out that loaded siRNA might get lost during the IP procedure due to dissociation from Ago2 and to unambiguously confirm that the IP procedure allows to quantitatively determine the amount of complex, we used 5’TMR-labelled single-stranded HuR siRNA complexed to recombinant Ago2 in vitro as a standard to quantify siRNA recovery by RT–qPCR, which yielded a relative recovery of Ago2–siRNA complexes in the protein IP of 83% (Supplementary Figure S2F). In addition, fluorescence anisotropy measurements confirmed that the recovered siRNA was still mostly complexed with Ago2 after native elution with the antigenic peptide (Supplementary Figure S2G). This data demonstrates the stability of the complex throughout the IP procedure, consistent with the long half-life of Ago2-guide strand complexes in vitro (half-life of Ago2-guide strand >20 h; Supplementary Figure S2H) and the high stability of the complex reported previously (Martinez and Tuschl, 2004), and altogether unambiguously confirm that the IP procedure allows to quantitatively determine Ago2-loaded siRNA within a range of ±20% accuracy. HeLa cells transfected with increasing doses of the very potent SSB siRNA reached saturation of the mRNA knockdown already at 0.25 nM transfected siRNAs, with an IC50 of 24±4 pM (Figure 2A). In contrast, the amount of loaded guide strand in Ago2 increased almost linearly up to the highest transfected dose (6.25 nM; Figure 2A, Supplementary Figure S2K and L). Consistent with observations emerging from previous in vivo studies (Pei et al, 2010), our data suggest that at siRNA concentrations beyond saturation of knockdown, a depot of loaded Ago2 may be formed and that the capacity of the RISC loading machinery is not yet saturated, at least for SSB siRNA concentrations of up to 200- to 300-fold beyond the IC50 (Figure 2A). Further calculations reveal that the IC50 of SSB mRNA knockdown is as little as 35–40 molecules of siRISC per cell (Figure 2B). To investigate whether these low numbers of siRISC per cell required for a 50% mRNA knockdown is a general feature of siRNAs, we performed the analogous experiments with absolute quantification for three additional siRNAs of markedly different potency (Supplementary Figure S2N–P). Two siRNAs were targeting other mRNAs (GAPDH and HuR), the third siRNA (‘SSB(53)’) targets another site within the same mRNA (SSB) but with significantly lower overall potency. Strikingly, for all of these siRNAs we find equally low numbers at IC50 ranging from 10 to 110 siRISC molecules per cell. These data suggest that RNA silencing is in general a remarkably efficient process once the siRNA is loaded into RISC. Additionally, we quantified the fraction of Ago2-loaded siRNA in relation to total intracellular siRNA which followed a hyperbolic saturation curve with increasing siRNA dose, with <1% fraction bound at the IC50 of knockdown and saturating at ca 4% maximal loading (Supplementary Figure S2I, J and M). This behaviour suggests that either compartmentalization or other limiting factors prevent a quantitative loading of the intracellular siRNA material into Ago2. Considering estimate accuracies of our correction for the efficiencies of all experimental steps characterized in detail above, this data suggest that even at maximum experimental underestimation, a major fraction of intracellular siRNA but also miRNA (see Figure 4B and Supplementary Figures S1H and S3B) is non-RISC associated, which may appear counter intuitive to the current assumption in the field. Interestingly however, a recent publication (Janas et al, 2012) reports a conclusion perfectly consistent with our quantitative data. Based on an absolute quantification of Ago proteins and total miRNA copies per cell, the authors come to the conclusion that there is a 13-fold excess of miRNA over Argonaute molecules in HeLa cells. This implies that only a few per cent of a given miRNA will be loaded in Ago proteins on average, in line with the data we show in Figure 4B and Supplementary Figures S1H and S3B. Figure 2.Excess of inactive siRNA masks active population. (A, B) Ago2 IPs were performed in a quantitative manner 24 h after the HeLa cells were transfected with SSB siRNA. mRNA levels were determined by RT–qPCR from purified total RNA. Download figure Download PowerPoint Active siRISC co-sediments with ER and Golgi membranes Given that such a minor fraction of intracellular siRNA gets loaded into Ago2, we concluded that for localization of siRNA activity, tracing bulk siRNA with microscopy or fractionation becomes very misleading. Therefore, we further refined the cell fractionation analysis of the siRNA by performing Ago2-IPs from continuous density sucrose gradients to quantify the amount of siRNA and miRNA loaded in Ago2 in each fraction. IP efficiencies were comparable across the gradient and well reproducible between independent fractionation experiments (Supplementary Figure S2D and E). Surprisingly and in contrast to the broad bulk distribution of siRNA, miRNA and RNAi pathway proteins, the Ago2-loaded SSB siRNA, miR-16 and miR-21 all eluted in a sharp peak in the Golgi and ER fractions (fractions 4–6; Figure 3A), suggesting that the active siRNA/miRNA population may be associated with Golgi and/or ER membranes. To get further evidence for activity of the siRNA-Ago2 complexes (siRISC) in these fractions, we next performed 5′RACE with RNA purified from each fraction to qualitatively assess the absence or presence of the cleavage product of the SSB mRNA. Consistent with the fractionation of the siRISC, a 5′RACE product was only detected in fractions 4–6 (Figure 3B). Sequencing of the 200 bp 5′RACE PCR product confirmed that all sequenced clones contained SSB mRNA cleaved at the expected position (Supplementary Figure S3A). Additionally, we tested the distribution of the cytoplasmatic 5′ to 3′ exonuclease Xrn1 in the same sucrose gradient fractions (Figure 1A). Xrn1 was present in all non-endo/lysosomal fractions and enriched in fractions 4–6. This shows that the sliced mRNA can even be detected in fractions where it can be degraded and in turn excludes the possibility that the absence of 5′RACE products in the fractions 1–3 and 7–9 was due to immediate mRNA degradation after the siRNA-mediated mRNA slicing. In summary, these experiments show that siRISC as well as the sliced mRNA do co-fractionate with Golgi and ER membranes. Figure 3.siRNA- and miRNA-loaded Ago2 complexes and mRNA slicing product co-sediment with ER/Golgi membranes. (A) Ago2 IPs were performed from fractions of the sucrose gradient displayed in Figure 1A, and SSB siRNA guide strand, miR-16 and miR-21 were quantified by RT–qPCR. (B) 5′RACE performed with total RNA from each of the sucrose gradient fractions of the gradient displayed in Figure 1A. PCR products were separated on an agarose gel, a positive 5′RACE signal results in a 208-bp fragment. Source Data for Figure 3 [embj201352-sup-0002-SourceData-S2.pdf] Download figure Download PowerPoint Active siRISC associates to the outside of the rER To investigate whether the active siRISC also physically associates with membranes, post-nuclear lysates of HeLa cells transfected with SSB siRNA were subjected to a membrane floatation assay on a discontinuous sucrose gradient (Tahbaz et al, 2004). After the centrifugation, membrane proteins of Lysosomes, Golgi and ER were enriched on the top (fraction 1 and partially 2), whereas non-membrane bound material remained in the loading zone (fractions 3–5; Figure 4A). Consistent with a previous report (Tahbaz et al, 2004), a portion of the cytoplasmatic population of Ago2 and Dicer, but also a fraction of Ago1 and PACT floated with membranes; Interestingly, TRBP was exclusively membrane associated. While the major siRNA, miRNA as well as Ago2 amounts were found in the non-membrane fractions, loaded siRISC as well as miRISC (miR-16 and miR-21) were strongly enriched in the membrane fractions (Figure 4B; Supplementary Figure S3B). Additionally, the SSB mRNA cleavage product floated exclusively with membranes (Figure 4C), whereas again, Xrn1 was present in all fractions (Figure 4A). Together, this suggested that the siRISC activity is not only co-fractionating with Golgi and ER membranes but is indeed membrane associated. To further characterize the Ago2 association with membranes, HeLa lysates were treated with RNAseA or ProteinaseK, and subsequently subjected to membrane floatation assays. RNAseA treatment did not lead to a detectable reduction in Ago2 in the membrane fraction (Figure 4D and Supplementary Figure S3C, corresponding non RNAse treated reference data in Figure 4A), indicating that indirect association via the target mRNA does not play a major role for Ago2 membrane association. Additionally, given that Ago2 membrane association is independent of RNA (Figure 4D), this rules out that Ago2 membrane association is mediated through polysomes. Figure 4.Active siRISC associates to the outside of membranes. HeLa cells were transfected with SSB siRNA and subjected to a membrane floatation assay. Fractions were analysed by western blotting (A), Ago2 IP and RT–qPCR (B) and 5′RACE (C) as in Figure 3. (D) HeLa lysates were treated with RNAseA or ProteinaseK and subjected to membrane floatation assays, fractions were analysed by western blotting. RNAse A activity was confirmed by analysis of GAPDH mRNA levels in the sucrose gradient fractions after the treatment (Supplementary Figure S3C). Source Data for Figure 4 [embj201352-sup-0003-SourceData-S3.pdf] Download figure Download PowerPoint Upon ProteinaseK treatment, all Ago2 was lost from the membrane fraction (Figure 4D). As controls, also Dcp1a which is not supposed to be encapsulated by membranes was fully susceptible to ProteinaseK treatment, whereas Calreticulin as an ER luminal protein was entirely protected from degradation and only detectable in the membrane fraction. In consequence and consistent with a previous report (Cikaluk et al, 1999), this data demonstrates that Ago2 as
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