In Vivo, Argonaute-Bound Micrornas Exist Predominantly in a Reservoir of Low Molecular Weight Complexes Not Associated with Mrna

In vivo, Argonaute-bound microRNAs exist predominantly in a reservoir of low molecular weight complexes not associated with mRNA

Gaspare La Roccaa,1, Scott H. Olejniczaka,1, Alvaro J. Gonzálezb, Daniel Briskinc, Joana A. Vidigala, Lee Spraggona, Raymond G. DeMatteoa, Megan R. Radlera, Tullia Lindstend, Andrea Venturaa, Thomas Tuschlc, Christina S. Leslieb, and Craig B. Thompsona,2

aCancer Biology and Genetics Program, bComputational Biology Program, and dImmunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and cHoward Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY 10065

Contributed by Craig B. Thompson, December 17, 2014 (sent for review September 30, 2014; reviewed by Joshua T. Mendell and Zissimos Mourelatos)

MicroRNAs repress mRNA translation by guiding Argonaute pro- (8, 9). These proteins bind one or more Argonaute proteins si- teins to partially complementary binding sites, primarily within multaneously and act as docking factors for the recruitment of the 3′ untranslated region (UTR) of target mRNAs. In cell lines, enzymes involved in translational repression and/or target deg- Argonaute-bound microRNAs exist mainly in high molecular weight radation (10). RNA-induced silencing complexes (HMW-RISC) associated with tar- Regulation of endogenous microRNA targets in mammals get mRNA. Here we demonstrate that most adult tissues contain occurs almost exclusively through the microRNA pathway, which reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) relies on assembly of large ribonucleoprotein complexes. As such, not bound to mRNA, suggesting that these microRNAs are not ac- Argonaute-bound microRNAs are found in distinct complexes, tively engaged in target repression. Consistent with this observa- ranging from low molecular weight RISC (LMW-RISC) com- tion, the majority of individual microRNAs in primary T cells were posed of an Ago protein complexed to an 18- to 25-nt microRNA ∼ enriched in LMW-RISC. During T-cell activation, signal transduction ( 100 kDa), to high molecular weight RISC (HMW-RISC), larger – through the phosphoinositide-3 kinase–RAC-alpha serine/threo- than 2 MDa (11 13). However, the biological significance of these nine-protein kinase–mechanistic target of rapamycin pathway in- complexes and what determines distribution of microRNAs be- creased the assembly of microRNAs into HMW-RISC, enhanced tween them is largely unknown. expression of the glycine-tryptophan protein of 182 kDa, an essential We have previously shown that accumulation of LMW-RISC component of HMW-RISC, and improved the ability of microRNAs can be induced in vitro by long-term depletion of nutrients or to repress partially complementary reporters, even when ex- growth factors from the medium of apoptosis-resistant cells (13). pression of targeting microRNAs did not increase. Overall, data Under these conditions, LMW-RISC is a stable complex that presented here demonstrate that microRNA-mediated target re- retains bound microRNAs, is able to reassemble into HMW-RISC, pression in nontransformed cells depends not only on abundance and can induce target repression upon restoration of growth conditions. These observations prompted us to examine the distribution of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling. Significance microRNA | Argonaute | GW182 | T cells | mTOR MicroRNAs limit gene expression by recruiting a large protein icroRNAs are ∼22 nucleotide (nt) single-stranded RNAs complex known as the RNA-induced silencing complex (RISC) to Mthat regulate gene expression by repressing translation of target mRNAs. While attempting to understand physiological target mRNAs and/or inducing their degradation (1). Because regulation of RISC assembly, we found that most healthy adult the majority of expressed mRNAs are predicted to be microRNA tissues retain a reserve of microRNAs not stably associated with target mRNA. Recruitment of microRNAs to large mRNA- targets (2), microRNA-mediated gene regulation is thought to containing complexes was accompanied by an increase in their contribute to most physiological and pathological processes. ability to repress targets and was regulated in part by phos- Nevertheless, elimination of an individual microRNA is rarely phoinositide-3 kinase–RAC-alpha serine/threonine-protein sufficient to trigger an overt phenotype in adult animals, yet fol- kinase–mechanistic target of rapamycin pathway-dependent lowing stress, phenotypes are often seen (3, 4), suggesting that enhancement of the glycine-tryptophan protein of 182 kDa microRNA utilization changes in response to external stimuli. protein expression. Data presented here suggest that in vivo, In mammals, most microRNAs are generated from long, RNA many expressed microRNAs exist in an inactive reserve, allowing polymerase II transcripts, which are reduced to an ∼22-nt double- resting cells to use microRNAs to dynamically regulate the stranded RNA fragment by two steps of cleavage, one medi- translation of target mRNAs in their environment. ated by the endoribonuclease Drosha in the nucleus and one by the endoribonuclease Dicer in the cytoplasm. One of the Author contributions: G.L.R., S.H.O., A.J.G., A.V., T.T., C.S.L., and C.B.T. designed research; two strands, known as the guide strand, is incorporated into G.L.R., S.H.O., D.B., J.A.V., L.S., R.G.D., M.R.R., and T.L. performed research; G.L.R., S.H.O., one of the four Argonaute proteins (Ago1–4), creating the A.J.G., D.B., J.A.V., T.L., A.V., T.T., C.S.L., and C.B.T. analyzed data; and G.L.R., S.H.O.,

minimal unit of the RNA-induced silencing complex (RISC) A.J.G., C.S.L., and C.B.T. wrote the paper. CELL BIOLOGY (5). Argonaute-bound microRNAs are capable of endonucleolytic Reviewers: J.T.M., University of Texas Southwestern Medical Center; and Z.M., University cleavage of perfectly complementary targets through the small of Pennsylvania. interfering RNA (siRNA) pathway (6) or translational repression The authors declare no conflict of interest. and degradation of mRNA targets with partial complementarity Data deposition: The sequencing datasets analyzed in this paper are available for down- through the microRNA pathway (7). Repression of partially load at cbio.mskcc.org/public/Leslie/PNAS2015_Thompson/. complementary target mRNAs requires interaction of microRNA- 1G.L.R. and S.H.O. contributed equally to this work. containing Argonaute proteins with one of three functionally 2To whom correspondence should be addressed. Email: [email protected]. redundant TNRC6 proteins, TNRC6A/the glycine-tryptophan This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. protein of 182 kDa, (hereafter GW182), TNRC6B, and TNRC6C 1073/pnas.1424217112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1424217112 PNAS | January 20, 2015 | vol. 112 | no. 3 | 767–772 Downloaded by guest on September 26, 2021 to assemble microRNAs into HMW-RISC and enhance target repression was regulated, at least in part, by induction of GW182 protein downstream of the phosphoinositide-3 kinase (PI3K)– RAC-alpha serine/threonine-protein kinase (Akt)–mechanistic target of rapamycin (mTOR) signaling pathway. Taken together, these data suggest that assembly of functional RISC is regulated globally by availability of RISC proteins and specifically by interactions between individual microRNAs and target mRNAs. Results RISC Molecular Weight Profile in Vivo. Assembly and function of the RISC has been extensively studied in immortalized cells grown in tissue culture (14). Our previous data demonstrated that RISC assembly and function could be modulated in cultured cells subjected to mitogen and/or nutrient deprivation and stimulation (13). Whether these observations could be extended to physiological, in vivo situations remained unknown. To investigate RISC assembly in vivo, we determined molecular weight profiles of Argonaute (Ago) proteins in tissues from healthy adult mice using Superose 6-based size exclusion chromatography. Strik- ingly, Ago proteins were greatly enriched in LMW-RISC in most adult tissues (Fig. 1A and SI Appendix, Figs. S1 and S2). In con- trast, Ago proteins were found almost exclusively in HMW-RISC in cell lines in log-phase growth, irrespective of species or tissue of origin (Fig. 1B and SI Appendix,Figs.S1andS2). This enrichment in HMW-RISC was not solely dependent on availability of Fig. 1. In vivo distribution of Ago2 protein between HMWR and LMWR. nutrients and mitogens in tissue culture medium because Ago Ago2 elution profiles determined by Superose 6 size exclusion chromatog- proteins were found enriched in HMW-RISC before in vitro dif- raphy followed by Western blot using extracts of (A) healthy adult mouse ferentiation of 3T3-L1 cells and in LMW-RISC following their tissues versus (B) cell lines in log-phase growth. Arrows indicate specific Ago2 differentiation to adipocytes (SI Appendix,Fig.S3). band, as a lower molecular weight nonspecific band was observed in some tissues. SI Appendix,Fig.S2provides quantification of replicate experiments. HMW-RISC Fractions Contain MicroRNA–Argonaute Complexes Stably Bound to mRNA. To better understand mechanisms that regulate distribution of Ago proteins between HMW-RISC and LMW- of microRNA–Argonaute complexes between HMW-RISC and RISC, we first examined biochemical properties that govern Ago LMW-RISC in vivo. We found that, whereas proliferating cells protein distribution in immortalized mouse embryonic fibro- maintain most microRNAs in HMW-RISC bound to mRNA, many blasts (MEFs). Consistent with HMW-RISC containing mRNA- adult tissues keep a large portion of their microRNA repertoire in bound Ago–microRNA complexes associated with polysomes (15) LMW-RISC, not engaged in target repression. Distribution be- and/or other mRNA binding proteins (16), Ago proteins coeluted tween LMW-RISC and HMW-RISC varied among microRNA with GW182, poly(A)-binding protein-1 (PABP1), and ribosomes species, as individual microRNAs showed characteristic dis- in high molecular weight fractions 8 and 9 (Fig. 2A). To test tributions between the two complexes. Furthermore, the ability this model, we assessed the requirement of RNA and protein

Fig. 2. HMW-RISC requires mRNA, microRNAs, and TNRC6 proteins. (A) Western blot analysis of proteins enriched in HMW-RISC fractions of Superose 6 elution profiles from MEFs in log-phase growth (Top). A total of 5 ng of recombinant GFP protein (spike-in) was spiked into each fraction to control for precipitation efficiency. Ethidium bromide staining of total RNA contained in each fraction separated on a 12% urea– PAGE gel is shown (Bottom). (B) Ago2 elution profile following RNaseA (2 μg/mL) treatment of lysates as in A.(C) Ago2 elution profile from extracts of Droshafl/fl MEFs stably expressing CreERT2 treated with 500 nM 4-hydroxytamoxifen (4-OHT) to induce Drosha de- letion or left untreated for 2 wk. (D) Western blot showing elution profile of Ago proteins in MEFs 48 h after cotransfection of siRNA against GW182, Tnrc6b, and Tnrc6c or control siRNA (siCTL). Western blots for polyA-binding protein 1 (PABP1) are shown to illustrate the specific effect on Ago proteins. (E) Repression of microRNA or siRNA reporter constructs by an oligonucleotide duplex transfected into MEFs as in D. Bars represent average reporter repression ±SD of three biological replicates (*P = 0.006 as determined by two-tailed Student’s t test).

768 | www.pnas.org/cgi/doi/10.1073/pnas.1424217112 La Rocca et al. Downloaded by guest on September 26, 2021 components of RISC for stable assembly of HMW-RISC. De- tissues (Fig. 5A compared with Fig. 1) as well as in several solid pletion of mRNA by treatment of extracts with RNaseA before tissues (SI Appendix, Fig. S10 compared with Fig. 1). fractionation led to complete dissolution of HMW-RISC (Fig. Consistent with an important role for GW182 in assembly of 2B). Similarly, knockout of Drosha (Fig. 2C and SI Appendix, Fig. HMW-RISC in hematopoietic cells, we found that increased S4) or Dicer (SI Appendix, Fig. S5) resulted in a marked re- GW182 expression following mitogenic stimulation was accompa- duction of HMW-RISC. nied by a shift of an exogenously introduced synthetic microRNA Additionally, siRNA-mediated depletion of all three TNRC6 from LMW-RISC to HMW-RISC and an increase in the ability proteins (GW182, TNRC6B, and TNRC6C) in MEFs led to of this synthetic microRNA to repress a reporter through the decreased HMW-RISC and accumulation of LMW-RISC (Fig. microRNA pathway (13) (SI Appendix, Fig. S11). Using the same 2D and SI Appendix, Fig. S6). Similar results were obtained using synthetic microRNA/reporter system, we found that microRNA the murine pro-B-cell line FL5.12. As TNRC6 proteins are function in activated T cells could be specifically inhibited by necessary and sufficient for microRNA-mediated target re- knockdown of GW182 (Fig. 5B and SI Appendix, Fig. S12), but pression (17), decreased HMW-RISC abundance upon knock- not by knockdown of TNRC6B or TNRC6C. This suggests that, down of all three TNRC6 proteins was accompanied by a among the three TNRC6 proteins, GW182 plays a critical role in E significant decrease in microRNA function (Fig. 2 ), as assessed enhanced microRNA function in hematopoietic tissues. by the ability of a transfected oligonucleotide duplex to repress a microRNA reporter construct. Interestingly, depletion of GW182 Expression, RISC Assembly, and MicroRNA Function Are TNRC6 proteins did not significantly affect repression of a Regulated by the PI3K-Akt-mTOR Signaling Pathway. To dissect siRNA reporter targeted by the same oligonucleotide duplex E the signaling responsible for enhanced GW182 expression, RISC (Fig. 2 ), supporting previous evidence that the microRNA assembly and microRNA function, freshly isolated T cells were and siRNA pathways are differentially dependent on TNRC6 proteins (10).

Distribution and Function Varies Between MicroRNA Species Independent of Their Expression. As mature microRNAs exist primarily bound to Ago proteins (18), distribution of Ago proteins between HMW- RISC and LMW-RISC should predict distribution of mature microRNAs. To test this hypothesis, we sequenced small RNAs contained in each Superose 6 fraction of lysates from freshly isolated, resting T cells that contain predominantly LMW-RISC and lysates from ex vivo stimulated T cells that contain predominantly HMW-RISC (Fig. 3A) (13). As expected, most microRNA species were enriched in LMW-RISC in resting T cells and in HMW- RISC in stimulated T cells (Fig. 3B and Dataset S1), regardless of their level of expression (SI Appendix,Fig.S7). Closer examination of these data revealed that redistribution to HMW-RISC following stimulation was distinctive for each microRNA, in agreement with previous reports (19). This shift from LMW-RISC to HMW- RISC did not significantly correlate with a change in microRNA expression (Fig. 3C and SI Appendix, Fig. S8). Analysis of individual microRNAs whose total expression did not change upon stimulation, but that redistributed between HMW-RISC and LMW-RISC, clearly demonstrated this principle (Fig. 3D). As expression and enrichment in target-bound RISC of several microRNA species were independent of one another, we hypoth- esized that function of these microRNAs may not be determined exclusively by their expression, but also by their enrichment in HMW-RISC. To test this hypothesis, the ability of two endogenous microRNA families to repress luciferase reporters was examined in resting and stimulated T cells. Expression of members of the miR- 15/16 seed family (miR-15a, miR-15b, and miR-16) decreased in stimulated T cells, yet showed a marked enhancement in HMW- RISC (Fig. 4A and SI Appendix,Fig.S9A) accompanied by enhanced repression of a reporter with target sites complementary to the miR-15/16 seed sequence (AGCAGCA; Fig. 4B). Similarly, expression of let-7 seed family microRNAs decreased following T-cell stimulation, whereas their HMW-RISC occupancy and ability to repress a reporter with target sites complementary to the Fig. 3. Distribution of microRNAs between HMW-RISC and LMW-RISC in let-7 seed sequence (GAGGUAG) increased (Fig. 4 C and D and resting and stimulated T cells. (A) Ago2 elution profile from freshly isolated SI Appendix,Fig.S9B). (resting) T cells versus T cells stimulated for 3 d with beads coated with αCD3 and αCD28 antibodies in the presence of rIL2 (25 units/mL). (B) Ratio of

GW182 Expression Predicts HMW-RISC Assembly and MicroRNA microRNAs found in HMW-RISC (fractions 8 and 9) to LMW-RISC (fractions CELL BIOLOGY 16–20) in T cells as in A. Bars represent each of the 110 microRNAs expressed Function in Hematopoietic Cells. Whereas differences existed be- in resting and/or stimulated T cells as determined by small RNA sequencing tween recruitment of individual microRNAs to HMW-RISC, from two independent experiments. (C) Change in expression of individual overall the trend was for increased occupancy in HMW-RISC of microRNA species (x axis) plotted against redistribution to HMW-RISC (y axis) both Ago proteins and microRNA following ex vivo stimulation following T-cell stimulation. Each dot represents a microRNA species. (D) of primary T cells. This enhancement of HMW-RISC assembly Elution profiles determined by small RNA sequencing of microRNA species

in stimulated T cells was accompanied by increased expression of whose expression did not change [false discovery rate, >10%, abs (log2 fold GW182 (13, 20) (Fig. 5A), one of three redundant RISC scaffold change) <0.5] upon stimulation illustrate a shift to HMW-RISC in the absence proteins. Expression of GW182 correlated with the extent to of expression change. The elution profiles of representative microRNAs which Ago protein partitioned to HMW-RISC in hematopoietic whose expression did not change with mitogenic stimulation are shown.

La Rocca et al. PNAS | January 20, 2015 | vol. 112 | no. 3 | 769 Downloaded by guest on September 26, 2021 to accumulation of Ago proteins in LMW-RISC (Fig. 6D) and decreased repression of reporters targeted by synthetic (Fig. 6E) or endogenous (SI Appendix, Fig. S15) microRNAs. Constitutive Akt activation was sufficient to maintain Ago protein occupancy in HMW-RISC (Fig. 6D) and microRNA reporter repression (Fig. 6E) in the absence of IL-3 stimulation. The mechanistic target of rapamycin (mTOR) is a central component of the PI3K-Akt signaling network known to in- fluence the fate of stimulated T cells (22). Inhibition of mTOR serine/threonine kinase activity using the chemical inhibitor Torin1 resulted in decreased expression of GW182 protein in stimulated T cells (Fig. 6F) or FL5.12 cells (SI Appendix, Fig. S16A). Furthermore, Torin1 treatment inhibited HMW-RISC assembly following T-cell stimulation (Fig. 6G) and decreased repression of a reporter construct targeted by an exogenously introduced synthetic microRNA in stimulated T cells (Fig. 6H)or FL5.12 cells (SI Appendix, Fig. S16B). In vivo, mTOR is a critical component of signal transduction-induced thymic T-cell matu- ration (23, 24) and development of the embryonic heart (25). Consistent with these observations, we found increased phosphorylation of the mTOR targets S6, S6 kinase, and 4E-BP1 when comparing freshly isolated thymocytes to mature T cells or embryonic heart to adult heart (Fig. 7A and SI Appendix, Fig. S17). Enhanced mTOR signaling correlated with increased GW182 protein expression (Fig. 7A) and Ago2 occupancy in HMW-RISC Fig. 4. Ability of endogenous microRNAs to repress reporters can be in- B C dependent of their expression. (A) Expression change plotted against dis- in both thymocytes (Fig. 7 ) and embryonic heart (Fig. 7 ). Taken tribution change of miR-15/16 seed family microRNAs (colored) following together, these data demonstrate that the relative abundance of ex vivo stimulation of T cells. Gray dots represent all other expressed HMW-RISC and LMW-RISC is dynamically regulated by the microRNAs. Change in expression of miR-15a/b and miR-16 was confirmed by PI3K-Akt-mTOR pathway, at least in part, through induction of qRT-PCR (SI Appendix,Fig.S9A). (B) Repression of a Firefly luciferase reporter GW182 expression. containing the miR-15/16 seed target sequence cloned into its 3′ UTR in resting and stimulated T cells. Bars represent average reporter repression ± Discussion SD of a representative experiment performed in quadruplicate (*P = 0.003). Altered expression of individual microRNAs has been linked to (C) Expression change plotted against distribution change of let-7 seed a variety of diseases, including cancer (26). As a consequence, family microRNAs (colored) following ex vivo stimulation of T cells. Gray dots extensive efforts have been directed to determine microRNA represent all other expressed microRNAs. Change in expression of let-7a and expression profiles (27). These studies tacitly assume that ex- let-7f were confirmed by qRT-PCR (SI Appendix, Fig. S9B). (D) Repression of pression level of a given microRNA correlates with its ongoing a Renilla luciferase reporter containing the let-7 seed target sequence cloned into its 3′ UTR in resting and stimulated T cells. Bars represent aver- repression of target mRNA. Data presented here show that age reporter repression ±SD of a representative experiment performed in differences in expression level of a given microRNA are not quadruplicate (*P = 0.005 as determined by two-tailed Student’s t test). necessarily predictive of the ability of that microRNA to repress its cognate targets. Rather, our data suggest that changes in intracellular signaling can alter microRNA assembly on targets, stimulated with microbeads coated with αCD3 and/or αCD28 and consequently function, independent of changes in expres- antibodies in the presence or absence of interleukin-2 (IL-2). sion. Moreover, microRNA–Argonaute complexes contained Maximal GW182 protein induction was seen following costi- in most adult tissues are not associated with target mRNA mulation with αCD3 and αCD28 for 24 h (Fig. 6A). As the (i.e., in HMW-RISC), suggesting that basal signaling required to phosphatidylinositol 3-kinase (PI3K) signaling pathway is a key mediator downstream of CD3/CD28 costimulation (21), the effect of the PI3K inhibitor LY294002 on GW182 protein expression and RISC composition was examined. As shown in Fig. 6 A and B, accumulation of GW182 protein and HMW-RISC in stimulated T cells could largely be prevented by the inhibitor, suggesting that signaling through the PI3K-Akt pathway stimulates HMW-RISC assembly through induction of GW182 expression. To confirm that the PI3K signaling pathway regulates HMW- RISC assembly, hematopoietic cell lines, in which mitogen- stimulated GW182 expression and HMW-RISC assembly are dependent on the presence of interleukin-3 (IL-3) (13), were used. Expression of tamoxifen-inducible PI3K P110 subunit (PI (3)K-ER) or myristoylated Akt (mAkt-ER) in BaF3 cells con- ferred 4-hydroxytamoxifen (4-OHT)-dependent GW182 protein expression in the absence of IL-3 (SI Appendix, Fig. S13). Simi- Fig. 5. Expression and function of GW182 in hematopoietic cells. (A)GW182 larly, constitutive expression of myristoylated Akt in FL5.12 cells expression in the indicated primary hematopoietic cell types assessed by was sufficient to maintain GW182 in the absence of IL-3, in- Western blot. FL5.12 cells expressing a control shRNA or a shRNA targeting GW182 mRNA are shown to demonstrate specificity of the GW182 antibody dependent of the antiapoptotic effect of constitutive PI3K-Akt used. (B) Repression of a microRNA reporter construct targeted by an oligo- pathway activation because GW182 expression was not main- nucleotide duplex (details in SI Appendix, SI Materials and Methods) transfected tained by overexpression of the antiapoptotic protein Bcl-xL into T cells expressing the indicated shRNAs and stimulated for 2 d with beads (Fig. 6C and SI Appendix, Fig. S14). Consistent with the putative coated with αCD3 and αCD28 antibodies in the presence of rIL2. Bars represent role of GW182 in HMW-RISC assembly and microRNA func- average reporter repression ±SD of a representative experiment performed in tion, IL-3 withdrawal from FL5.12 cells expressing Bcl-xL led quadruplicate (*P = 0.003 as determined by two-tailed Student’s t test).

770 | www.pnas.org/cgi/doi/10.1073/pnas.1424217112 La Rocca et al. Downloaded by guest on September 26, 2021 proteins have slicing activity that can degrade exogenous nucleic acids complementary to bound nucleic acids. Although metazoan microRNAs are thought to regulate gene expression primarily through translational repression, the structural conservation between eukaryotic Ago2 and its archeal and eubacterial counter- parts is striking (31). Our data show that in conditions where LMW-RISC predominates, microRNA activity is compromised but siRNA activity is not (Figs. 2E,6E, and SI Appendix, Fig. S15B). These observations raise the possibility that microRNAs stored in LMW-RISC may not only be recruited to HMW-RISC to regulate endogenous gene expression, but may also be used to cope with exogenous nucleic acids introduced by pathogens. MicroRNA function can be regulated at the level of RISC assembly via regulation of GW182 protein expression. When comparing distribution of individual microRNAs between HMW-RISC and LMW-RISC in conditions where GW182 is not limiting (i.e., stimulated T cells), we observed differences among microRNA species. This suggests that association of individual microRNAs with target mRNAs is further regulated by factors that restrict or enhance these interactions. It is likely that differential activation of target mRNAs also contributes to differences in distribution of individual microRNAs between HMW-RISC and LMW-RISC during mitogenic activation. In future studies, the mapping of microRNA-target interactions during physiologic perturbations of primary tissues using methods such as high-throughput sequencing Fig. 6. PI3K-Akt-mTOR signaling promotes GW182 expression, HMW-RISC of RNA isolated by cross-linking immunoprecipitation or photo- assembly, and microRNA function. (A) GW182 expression by Western blot in activatable-ribonucleoside-enhanced cross-linking and immuno- T cells stimulated for 24 h with bead-bound αCD3 antibody, αCD28 antibody, precipitation may help shed light on these issues. Furthermore, or rIL-2 (25 units/mL) alone or in combination ± the PI3K inhibitor LY294002 recent studies have reported additional ways in which microRNA– (10 μM). (B) Ago2 elution profile from T cells treated as in the last two lanes mRNA interactions can be altered, including changes in sub- of A.(C) Western blot for GW182 expression in IL-3 dependent FL5.12 cells cellular localization of microRNA–Ago complexes (32–34), ex- engineered to overexpress Bcl-xL (FL5.12.xL) or myristoylated Akt (FL5.12. pression of endogenous microRNA “sponges” (35), microRNA mAkt) cultured in the presence or absence of IL-3 for 24 h. (D) Comparison of Ago2 elution profiles from FL5.12.xL versus FL5.12.mAkt cells cultured in the editing (36), interference by RNA-binding proteins (16, 37), presence or absence of IL-3 for 72 h. (E) Repression of microRNA or siRNA changes in RNA secondary structures (38), use of alternative reporter constructs by an oligonucleotide duplex transfected into cells cul- polyadenylation sites in target mRNAs (39), or disruption of tured as in C. Bars represent average reporter repression ±SD of a repre- microRNA–Ago interactions (40). All these factors collectively sentative experiment performed in quadruplicate (*P < 0.001). (F) Western blot for GW182 expression in T cells stimulated with beads coated with αCD3 and αCD28 antibodies in the presence of rIL2 ± 500 nM Torin1 for 48 h. (G) Ago2 elution profile from T cells treated as in the last two lanes of F.(H) Repression of a microRNA reporter construct by an oligonucleotide duplex transfected into T cells cultured as in the last two lanes of F. Bars represent average reporter repression ±SD of a representative experiment performed in quadruplicate (*P < 0.001 as determined by two-tailed Student’s t test).

maintain tissue homeostasis is insufficient to promote microRNA function. In light of these observations, it follows that lack of apparent phenotypes observed in many microRNA knockout animals (3, 4) may be, at least partially, explained by the fact that microRNAs are not associated with target mRNA in most differentiated adult tissues. The endpoint of the microRNA pathway is mRNA degradation (28), a function apparently redundant to the siRNA pathway. The siRNA pathway requires, in principle, only the catalytic activity of Ago2 to induce target degradation (29), whereas the microRNA pathway requires recruitment of proteins responsible for deadenylation and degradation of mRNA targets through association of target-bound Argonaute proteins with GW proteins (GW182, TNRC6B, and TNRC6C). Data presented here suggest that regulation of this interaction, and therefore the CELL BIOLOGY assembly of a functional RISC, may allow cells to express Fig. 7. In vivo correlation between mTOR signaling pathway activation, microRNAs in advance of their physiologic requirement. This GW182 protein expression, and enrichment of Ago proteins in HMW-RISC. may provide an advantage during transition between physio- (A) Western blot for phosphorylation of mTOR pathway activation and logical states or during stress, which could help to explain why GW182 protein expression in the indicated tissues or cell types. (B) Ago2 metazoans evolved the microRNA pathway while maintaining elution profiles determined by Superose 6 size exclusion chromatography the siRNA pathway. followed by Western blot from freshly isolated thymocytes versus resting Recently, it was reported that bacterial Ago proteins can re- splenic T cells. (C) Ago2 elution profiles determined by Superose 6 size ex- strict the acquisition of foreign nucleic acids using an siRNA- clusion chromatography followed by Western blot from embryonic heart related process (30). Like mammalian Ago2, the bacterial Ago (E13.5) versus adult heart. Arrow indicates specific Ago2 band.

La Rocca et al. PNAS | January 20, 2015 | vol. 112 | no. 3 | 771 Downloaded by guest on September 26, 2021 may contribute to the distribution of individual microRNAs be- Materials and Methods tween HMW-RISC and LMW-RISC in vivo. T-Cell Stimulation. Splenic T cells were isolated from 6- to 12-wk-old C57/BL6 Previous studies, such as those describing the creation of tar- mice and activated using antibody-coated microbeads in the presence or get prediction algorithms, have focused on cell lines (41). Here absence of recombinant mouse IL-2 (rIL-2, Becton Dickinson). we demonstrate that in cultured cell lines, the vast majority of expressed microRNAs are found in HMW-RISC, regardless of Size Exclusion Chromatography and Assessment of MicroRNA Distribution their expression level. Because HMW-RISC engages in target Profiles. Crude cell lysate was processed through Superose 6 column (GE repression, this explains why there is such tight correlation be- Healthcare) loaded on an AKTA FPLC system (GE Healthcare). Small RNA was tween microRNA expression and target repression in cell lines. isolated from each Superose 6 fraction by miRNeasy Serum/Plasma Kit Whereas this correlation has allowed the effective development (QIAGEN). Next, RNA was ligated to a barcoded 3′ adapter, pooled, ligated to of algorithms to predict microRNA targets, the study of freshly a common 5′ adapter, reverse transcribed into cDNA, PCR amplified, and isolated adult tissue reveals an additional level of complexity sequenced on the Illumina HiSeq platform. to how microRNAs function in vivo. Adult tissues contain high levels of microRNA/Ago complexes that exist in LMW complexes MicroRNA Reporters. MicroRNA function was tested using Dual-Luciferase Reporter System (Promega). MicroRNA reporters containing specific target that are not engaged in target repression. Such LMW-RISC may ′ represent a regulatory reserve that is used in vivo to allow cells sequences at the 3 end of luciferase genes were cotransfected with control reporters to normalize transfection efficiency. to modulate gene expression in response to physiologic pertur- A detailed description of methods and reagents can be found in SI Appendix, bations or in response to stress. As a corollary, it is likely that the SI Materials and Methods. distribution of microRNAs between HMW-RISC and LMW-RISC can provide additional information concerning the contribution ACKNOWLEDGMENTS. We thank Chao Lu (The Rockefeller University) for of individual microRNAs to gene regulation during physiological technical assistance with the 3T3 L1 differentiation assay; Jayanta Chaudhuri and/or pathological conditions. Further analysis will clarify for sharing the AKTA FPLC system; and Christine Mayr, Raymond Wu, whether microRNA distribution is predictive of which microRNAs Julija Hmeljak, Ping Mu, and members of the C.B.T. laboratory for critical are engaged in translational repression. If so, such analyses may discussions and feedback on the manuscript. This work has been funded by the National Institutes of Health Grants R01 CA105463 (to C.B.T.), K99 provide a tool to generate microRNA utilization profiles, which CA175189 (to S.H.O.), R01 CA149707 GRANT10355609 (to A.V.), U01-CA164190 will complement the ongoing analysis of microRNA by expres- and U24-CA143840 (to C.S.L.), and Memorial Sloan-Kettering Cancer Center sion profiling. Support Grant P30 CA008748.

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