Ribosomal Protein L11 Recruits miR-24/miRISC To Repress c-Myc Expression in Response to Ribosomal Stress ^▿

Cell culture, antibodies, plasmids, and reagents.

Human lung non-small cell carcinoma H1299 cells, human osteosarcoma U2OS cells, and human embryonic kidney epithelial 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 U of penicillin/ml, and 0.1 mg of streptomycin/ml at 37°C in a 5% CO₂ humidified atmosphere (18). Human diploid lung fibroblast WI38 cells were cultured in DMEM supplemented with 15% FBS and MEM nonessential amino acids (Gibco). To observe the c-Myc regulation by overexpression of L11, cells were transfected and cultured in DMEM containing 0.2% FBS. Anti-Flag (M2; Sigma), rabbit polyclonal anti-Ago2 (Millipore), mouse monoclonal anti-Ago2 (Abcam), mouse monoclonal anti-Myc (9E10; Zymed), and mouse polyclonal anti-Myc (Y69; Abcam) antibodies were purchased. Rabbit polyclonal anti-L11 (12) and anti-L23 (18) antibodies were previously described. Act D and 5-FU were purchased from Sigma. The Flag-tagged L11 (Flag-L11) plasmid was previously described (12). To construct a pGL3-myc3′UTR luciferase reporter, the c-myc 3′-UTR was amplified from U2OS cell mRNA by reverse transcriptase PCR (RT-PCR) with primers 5′-CGCTCTAGAGGAAAAGTAAGGAAAACGATTCCTTC-3′ and 5′-CGCTCTAGATTGGCTCAATGATATATTTGCCAG-3′, where the underlined sequences represent the XbaI site. The PCR product was then cloned into pGL3-promoter plasmid (Promega) at the XbaI site and sequenced. The luciferase reporters containing different fragments of c-myc 3′-UTR were cloned by inserting PCR products into the pGL3-promoter plasmid at the XbaI site.

Transfection, immunoblot, and coimmunoprecipitation analyses.

Cells were transfected with plasmids by the use of TransIT-LT1 reagents following the manufacturer's protocol (Mirus Bio Corporation). Cells were harvested at 48 h posttransfection and lysed in lysis buffer consisting of 50 mM Tris-HCl (pH 8.0), 0.5% Nonidet P-40, 5 mM EDTA, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 1 μg of pepstatin A/ml, and 1 mM leupeptin. Equal amounts of cell lysates were used for immunoblot (IB) analysis as described previously (16). Coimmunoprecipitation (co-IP) was conducted as described previously (14, 16). Sequential co-IP of L11 and c-Myc was conducted as described previously (12, 17). RNA was extracted from the immunoprecipitates and subjected to RT-quantitative PCR (RT-qPCR) assays as described below. For analysis of the RNA dependency of L11-Ago2 interaction (see Fig. 3), RNase A (200 μg/ml) was added to cell lysates followed by IP.

[³⁵S]methionine pulse-labeling.

To examine c-Myc translation upon L11 knockdown, cells were prestarved in methionine-free medium supplemented with dialyzed 10% FBS for 30 min followed by pulse-labeling with 50 μCi of [³⁵S]methionine/ml for 15 min. Cells were lysed, and the cleared cell lysates were immunoprecipitated using anti-c-Myc (C33; Santa Cruz Biotechnology) monoclonal antibody-conjugated beads. After washing, half of the immunoprecipitate was separated using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The gel was incubated in Amplify solution (Amersham Biosciences) for 10 min, dried, and exposed to X-ray film. The other half was subjected to IB using anti-c-Myc (Y69) antibodies. Radiolabeled and total c-Myc were quantified, and the ratio of radiolabeled to total c-Myc represents the relative translational activity of c-Myc. Equal amounts of total proteins were also loaded onto an SDS-PAGE gel to examine total ³⁵S-labeled cellular proteins.

Immunoprecipitation of protein-associated RNAs (RNA IP).

Immunoprecipitation of RNA-protein complexes was performed as previously described, with minor modifications (37, 60). Briefly, cells were lysed in polysome lysis buffer (PLB) (100 mM KCl, 5 mM MgCl₂, 10 mM HEPES [pH 7.0], 0.5% Nonidet P-40, 1 mM DTT, 100 U of RNase inhibitor/ml) supplemented with 20 mM EDTA and protease inhibitors on ice for 20 min followed by centrifugation. The supernatants were precleared with protein A-Sepharose beads. The cleared supernatants were then diluted (1:10 [vol/vol]) in NT2 buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1 mM MgCl₂, 0.05% Nonidet P-40, 1 mM DTT, 100 U of RNase inhibitor/ml) supplemented with 20 mM EDTA and protease inhibitors and incubated with primary antibodies at 4°C for 4 h, followed by incubation of protein A/G beads for an additional 2 h at 4°C. The beads were washed five times with NT2 buffer supplemented with protease inhibitors. The bead-bound protein-RNA complexes were then treated with DNase I and proteinase K and eluted twice with NT2 buffer containing 0.1% SDS. RNAs were extracted from the elution with phenol-chloroform and ethanol precipitation and subjected to RT-qPCR assays as described below.

Luciferase reporter assays.

Cells were transfected with pCMV–β-galactoside (β-gal) and luciferase reporter plasmid pGL3, with pGL3-myc3′UTR or its fragments, together with control or Flag-L11 plasmid, or with scrambled or L11 small interfering RNA (siRNA), as indicated in the figures. Luciferase activity was determined and normalized by calculating β-gal activity in the same assay as described previously (12).

RT-qPCR analysis.

Total RNA was isolated from cells by the use of TRIzol reagent (Invitrogen). Reverse transcriptions were performed as described previously (59). qPCR was performed using an ABI StepOne real-time PCR system (Applied Biosystems) and SYBR green mix (Bio-Rad) for mRNA expression determinations as described previously (59). Analysis of expression of mature miRNAs was performed using a TaqMan miRNA assay kit (Applied Biosystems) following the manufacturer's protocol. All reactions were carried out in triplicate. Relative gene expression levels were calculated using the ΔCτ method following the manufacturer's instructions. The primers for c-myc and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were previously described (12). The primers for other genes were 5′-CATCGTTGACCGCCTGAAGT-3′ and 5′-GGAGCAAGATGGATTCCAATTC-3′ (for luciferase); 5′-TCAACGCGCAGGACTTCTG-3′ and 5′-CAGTGACCGTGGGAATGAAGTT-3′ (c-fos); 5′-GCAAAGATGGAAACGACCTTCT-3′ and 5′-GCTCTCGGACGGGAGGAA-3′ (c-jun); 5′-GCCTGCGACATCTGTGGAA-3′ and 5′-CGCAAGTGGATCTTGGTATGC-3′ (egr1); 5′-AGGCCTTGGAACTCAAGGAT-3′ and 5′-TGAGTCAGGCCCTTCTGTCT-3′ (p53); 5′-ATGAATCCCCCCCTTCCAT-3′ and 5′-CAGGAAGCCAATTCTCACGAA-3′ (mdm2); and 5′-GCCTTGAGGAAGGATTGGTA-3′ and 5′-TCGACAATCAGGGACATCAT-3′ (mdmx).

RNA interference (RNAi) and miRNA overexpression.

The 21-nt siRNA duplexes with a 3′ dTdT overhang were synthesized by Dharmacon Inc. (Lafayette, CO). The target sequences for L11 and control scramble II RNA were previously described (12). L11 siRNA-1 was used in all the experiments except where indicated. The target sequence for Ago2 was 5′-GCACGGAAGUCCAUCUGAA-3′. The miR-24 mimics and control cel-miR-67 were purchased from Dharmacon Inc. These siRNA duplexes (100 nM) and miRNA mimics (25 to 50 nM) were introduced into cells by the use of SilentFect lipid reagent (Bio-Rad) following the manufacturer's protocol. The cells were analyzed 48 h after transfection.

Cell fractionations.

To isolate the cytoplasmic and nuclear fractions, cells were resuspended in hypotonic buffer A (10 mM HEPES [pH 7.9], 10 mM KCl, 1.5 mM MgCl₂, 0.5 mM DTT) in the presence of protease inhibitors. Cell membranes were then broken using a Dounce homogenizer (tight pestle) and 10 up-and-down strokes. After centrifugation, the supernatant was collected as the cytoplasmic fraction and supplemented with a 0.1 volume of 10× buffer B (0.3 M Tris-HCl [pH 7.9], 1.4 M KCl, 30 mM MgCl₂). The nuclear pellets were washed with buffer A and then resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 0.2 mM EDTA, 1.5 mM MgCl₂, 0.5 mM DTT, 25% glycerol) in the presence of protease inhibitors and sonicated. The nuclear fraction (supernatant) was collected by centrifugation.

For isolation of the nucleolus fraction, the nuclear pellets were resuspended in buffer S1 containing 0.25 M sucrose and 10 mM MgCl₂, layered over buffer S2 containing 0.35 M sucrose and 0.5 mM MgCl₂, and centrifuged at 1,430 × g for 10 min at 4°C. The pelleted nuclei were resuspended in buffer S2 followed by sonication. The sonicated nuclei were then layered over buffer S3 containing 0.88 M sucrose and 0.5 mM MgCl₂ and centrifuged at 3,000 × g for 10 min at 4°C. The pellet contained purified nucleoli, and the supernatant represented the nucleoplasm (2, 4).

Ribosome fractionation.

For separation of ribosome-free L11 from the total ribosome volume, cells were lysed in polysome lysis buffer. The extracts were overlaid on a 20% (wt/vol) sucrose cushion and centrifuged at 150,000 × g for 2 h. The polysome-containing pellet and the nonribosomal supernatant were collected separately (42) and immunoblotted with anti-L11 antibody. The nonribosomal supernatant was also used for RNA IP assays.

L11 regulates the levels and stability of c-myc mRNA.

We previously showed that L11 directly suppresses c-Myc transactivation activity (12). Interestingly, L11 also regulates c-Myc levels. Knockdown of L11 drastically increased the levels of c-Myc protein (12) (bottom panels of Fig. 1 A) and c-myc mRNA (15) in U2OS cells. RT-qPCR assays revealed that the levels of c-myc mRNA were markedly increased upon knockdown of L11 (top panel of Fig. 1A). This effect was unlikely to have been an off-target effect, as similar results were observed when two different siRNAs against L11 were used. To examine how c-myc mRNA was induced by knockdown of L11, we determined the half-life (t_1/2) of c-myc mRNA. As shown in Fig. 1B, knockdown of L11 significantly prolonged the half-life of c-myc mRNA, suggesting that knockdown of L11 stabilizes c-myc mRNA. To examine whether knockdown of L11 affects c-Myc translation, we performed [³⁵S]methionine pulse-labeling assays. Consistent with a previous report (4), transient knockdown of L11 did not significantly affect global translation (left panel of Fig. 1C). It also appears that knockdown of L11 did not affect c-Myc translation, as levels of nascent synthesized c-Myc were increased proportionally to total cellular c-Myc levels (right panels of Fig. 1C). Therefore, we conclude that transient knockdown of L11 increases c-Myc levels by stabilizing c-myc mRNA without affecting c-Myc translation. Consistently, overexpression of L11 reduced the levels of c-myc mRNA and protein (Fig. 1D) in U2OS cells. Similarly, knockdown of L11 also increased the levels of c-myc mRNA and protein in 293 cells (Fig. 1E), human normal fibroblast WI38 cells (Fig. 1F), and H1299 cells (data not shown), suggesting that the regulation of c-myc mRNA by L11 was not specific to cell type. Taken together, these results demonstrate that L11 regulates c-myc mRNA turnover.

L11 binds to c-myc at its 3′-UTR.

To understand how L11 regulates c-myc mRNA stability, we examined whether L11 physically associates with c-myc mRNA by the use of RNA immunoprecipitation (IP) assays. Lysates from 293 or U2OS cells transfected with Flag-L11 were immunoprecipitated with control IgG or anti-Flag antibody, followed by detection of c-myc mRNA by the use of RT-qPCR assays. As shown in Fig. 2 A, c-myc mRNA was specifically detected in anti-Flag but not control IgG immunoprecipitates from both cell lines, suggesting that L11 binds to c-myc mRNA in cells.

c-myc mRNA turnover is fast and highly regulated in cells, with a normal half-life of about 15 to 30 min (19) (Fig. 1B). The c-myc 3′-UTR contains several AU-rich elements (AREs) and plays an important role in the regulation of c-myc mRNA turnover (30). Several ARE binding proteins, including AUF1 (67), HuR (32), and tristetraprolin (TTP) (39), have been found to bind to c-myc AREs and regulate c-myc mRNA stability. To test whether L11 binds to c-myc mRNA through its 3′-UTR, we generated a luciferase reporter that contained a c-myc 3′-UTR in the 3′ portion of the luciferase gene (pGL3-myc3′UTR; Fig. 2E). U2OS cells were transfected with Flag-L11 together with pGL3-myc3′UTR or control pGL-3 vector. The cell lysates were immunoprecipitated with anti-Flag antibody or control IgG followed by RT-qPCR detection of the luciferase gene. As shown in Fig. 2B, luciferase mRNA was specifically coimmunoprecipitated with Flag-L11 in cells transfected with c-myc 3′-UTR-containing but not control luciferase plasmid. Similar results were also observed in 293 cells (Fig. 2F; see also Fig. 8). These results demonstrate that L11 binds to the c-myc 3′-UTR in cells.

L11 regulates c-myc mRNA levels through the c-myc 3′-UTR.

Next, we examined whether L11 regulates c-myc mRNA levels through the c-myc 3′-UTR. 293 cells were transfected with pGL3 or pGL3-myc3′UTR together with control or Flag-L11 plasmid. The cells were assayed for relative luciferase activity levels as well as expression of luciferase mRNA by RT-qPCR. As shown in Fig. 2C, overexpression of L11 specifically suppressed the luciferase activity (top panel) and reduced luciferase mRNA levels (middle panel) in pGL3-myc3′UTR but not control pGL3 transfected cells. Similar results were also observed in H1299 cells (data not shown). Conversely, knockdown of endogenous L11 significantly increased the luciferase activity and the luciferase mRNA levels in pGL3-myc3′UTR (compare lane 4 to lane 3) but not control pGL3 (compare lane 2 to lane 1) transfected U2OS cells (Fig. 2D). Together, these results suggest that L11 regulates c-myc mRNA levels by acting on the c-myc 3′-UTR.

To demonstrate the specificity of L11 regulation of c-myc mRNA through the c-myc 3′-UTR, we constructed a panel of luciferase reporter plasmids containing overlapping fragments of the c-myc 3′-UTR (Fig. 2E) and mapped the L11-binding site at the c-myc 3′-UTR. 293 cells were transfected with Flag-L11 together with control pGL-3 and pGL3-myc3′UTR or its deletion fragments followed by RNA IP using control IgG or anti-Flag antibodies. As shown in Fig. 2F, Flag-L11 was associated specifically with the luciferase mRNA containing the full-length (FL) c-myc 3′-UTR and the fragment consisting of nt 309 to 470 (F4) but not other fragments encompassing nt 1 to 360. Furthermore, overexpression of L11 significantly suppressed the luciferase activity in cells transfected with pGL3-myc3′UTR-F4 but not those transfected with other c-myc 3′-UTR fragments containing luciferase reporter plasmids (F1, F2, and F3) (Fig. 2G). These results suggest that L11 regulates c-myc mRNA through nt 361 to 470 of the c-myc 3′-UTR.

L11-mediated c-myc mRNA reduction requires Ago2.

miRNAs have emerged as key regulators of gene expression by suppressing translation and/or inducing mRNA decay of target genes through the 3′-UTR (47, 61). Therefore, we asked whether L11 regulates c-myc mRNA turnover through a miRNA-mediated pathway. As Ago2 is a core component of miRISC (47, 61) and, consistent with a previous report (32), knockdown of endogenous Ago2 significantly increased the levels of c-Myc protein and mRNA (Fig. 3 A), we tested whether Ago2 is involved in L11-mediated c-myc mRNA reduction. We first asked whether L11 could facilitate the recruitment of Ago2 to the c-myc mRNA. To this end, we performed RNA IP followed by RT-qPCR assays and found that overexpression of L11 increased the binding of Ago2 to the c-myc mRNA (Fig. 3B), whereas knockdown of L11 significantly reduced the Ago2 binding to the c-myc mRNA (Fig. 3C), suggesting that L11 facilitates the recruitment of Ago2 to the c-myc mRNA. Furthermore, knockdown of Ago2 extensively rescued the L11-mediated reduction of c-Myc protein (compare the ratio of lane 4 to lane 3 with the ratio of lane 2 to lane 1 in Fig. 3D) and c-myc mRNA (Fig. 3E) in U2OS cells. Together, these results suggest that L11 decreases c-myc mRNA levels by recruiting Ago2 to the c-myc mRNA.

L11 associates with Ago2.

Given that L11 regulates Ago2 targeting of c-myc mRNA (Fig. 3B and C), we tested whether L11 physically associates with Ago2. 293 cells were transfected with control or Flag-L11 followed by co-IP assays using anti-Flag antibodies. As shown in Fig. 3F, endogenous Ago2 was specifically coimmunoprecipitated with Flag-L11 in 293 cells. Similar results were also observed in U2OS cells (Fig. 3G) and H1299 cells (data not shown). Also, endogenous Ago2 and endogenous L11 were specifically coimmunoprecipitated with each other using either anti-Ago2 or anti-L11 antibodies, but not control IgG, in 293 cells (Fig. 3H) and U2OS cells (Fig. 3I and data not shown). RNase treatment abolished the interaction between L11 and Ago2 (Fig. 3I). These results suggest that the interaction between L11 and Ago2 in cells is dependent on the presence of cellular RNA.

L11 recruits miR-24 to target c-myc mRNA.

We then asked whether L11 regulates c-myc mRNA through the activity of miRNAs. At least four miRNAs, including Let-7 (32), miR-145 (55), miR-24 (34), and miR-34c (7), have been previously shown to downregulate c-Myc by either repressing c-Myc translation (let-7 and miR-34c) or reducing c-myc mRNA levels (miR-24 and miR-145). Thus, we examined whether L11 associates with these miRNAs in cells. Lysates from 293 or U2OS cells transfected with Flag-L11 were immunoprecipitated with anti-Flag antibodies or control IgG followed by RT-qPCR detection of the miRNAs described above. U6 snRNA was used as a control. As shown in Fig. 4 A, miR-24, but not let-7b, miR-34c, and U6 snRNA, was significantly enriched in the Flag-L11 immunoprecipitates compared to control IgG in 293 cells. Similar results were also observed in U2OS cells except that miR-34c was undetectable in the immunoprecipitates (Fig. 4B). We were unable to detect miR-145 in the immunoprecipitates from both 293 and U2OS cells (data not shown). These results clearly indicate that L11 specifically associates with miR-24 but not with other known c-myc targeting miRNAs.

Next, we examined the role of miR-24 in L11-mediated c-myc mRNA decay. Consistent with a previous report (34), overexpression of miR-24 significantly reduced the levels of c-Myc protein and mRNA in a dose-dependent manner (Fig. 4C). We then transfected U2OS cells with different combinations of scrambled or L11 siRNA and control (cel-miR-67) or miR-24 mimics. The cells were analyzed for expression of c-Myc protein and mRNA. Interestingly, knockdown of L11 drastically rescued the miR-24-mediated reduction of c-Myc protein levels (compare the ratio of lane 4 to lane 3 with the ratio of lane 2 to lane 1; Fig. 4D) and c-myc mRNA levels (Fig. 4E). Similarly, knockdown of L11 significantly abolished the miR-24-mediated reduction of relative luciferase activity in cells transfected with pGL3-myc3′UTR reporter plasmid (Fig. 4F). These results suggest that L11 plays a critical role in miR-24-mediated downregulation of c-myc mRNA, supporting the notion that L11 regulates c-myc mRNA by recruiting miR-24-loaded miRISC to the c-myc 3′-UTR.

c-myc mRNA is reduced in response to ribosomal stress.

To examine the physiological relevance of the L11 regulation of c-myc mRNA turnover, we asked whether L11 is involved in c-myc regulation in cells in response to ribosomal stress, as L11 plays a key role in p53-mediated cell cycle control in response to such stress (4, 36, 57–59, 69). Ribosomal stress is triggered following perturbation of ribosomal biogenesis, including inhibition of rRNA synthesis by treatment with a low dose of Act D (5 nM) (4, 14, 18) and inhibition of rRNA processing by treatment with 5-FU (57). c-Myc has recently been shown to be downregulated in response to DNA damage (7, 48). To test whether c-Myc levels are also downregulated by ribosomal stress, we treated U2OS cells with a low dose of either Act D or 5-FU followed by the detection of c-Myc expression. As shown in Fig. 5 A, treatment of cells with either Act D or 5-FU significantly reduced the c-Myc protein levels in a time-dependent manner. This reduction correlates with the reduction of c-myc mRNA levels (Fig. 5B), suggesting that ribosomal stress induces c-myc mRNA degradation.

L11 and Ago2 are required for c-myc mRNA downregulation in response to ribosomal stress.

Next, we asked whether L11 is involved in c-Myc downregulation in response to ribosomal stress. U2OS cells were transfected with scrambled or L11 siRNA followed by treatment with control dimethyl sulfoxide (DMSO), Act D, or 5-FU. As shown in Fig. 5C, knockdown of L11 completely abolished the c-Myc protein reduction caused by the Act D or 5-FU treatment. Again, this occurred at the mRNA level, as knockdown of L11 rescued the reduction of c-myc mRNA levels caused by Act D or 5-FU treatment (Fig. 5D). To evaluate the involvement of miRNA-mediated silencing in the downregulation of c-myc mRNA by ribosomal stress, we performed Ago2 knockdown experiments. Indeed, knockdown of Ago2 significantly rescued the downregulation of c-Myc protein (Fig. 5E) and mRNA (Fig. 5F) levels caused by treatment with Act D or 5-FU. Thus, L11 and Ago2 are required for c-myc mRNA downregulation by ribosomal stress.

L11 recruits miR-24-loaded miRISC to c-myc mRNA in response to ribosomal stress.

As L11 has been previously shown to dissociate from intact ribosomes into the ribosome-free pool (free L11) in response to ribosomal stress (4, 57), we reasoned that free L11 might be able to associate with c-myc mRNA. Thus, U2OS cells treated with Act D, 5-FU, or DMSO were subjected to RNA IP using anti-L11 antibodies, followed by RT-qPCR assays. As shown in Fig. 6 A, L11 binding to c-myc mRNA significantly increased in cells treated with Act D or 5-FU compared to the control results, indicating that ribosomal stress enhances L11 targeting of c-myc mRNA.

Next, we examined whether L11 recruits miRISC to c-myc mRNA in response to ribosomal stress. The cell lysates from U2OS cells treated with DMSO, Act D, or 5-FU were immunoprecipitated with anti-Ago2 antibodies or control IgG. As shown in Fig. 6B, treatment of cells with Act D or 5-FU markedly increased the association of Ago2 with c-myc mRNA. This increase requires L11, as knockdown of L11 significantly reduced the binding of Ago2 with c-myc mRNA in cells treated with Act D or 5-FU (Fig. 6C). To confirm that L11 interacts with Ago2 under conditions of ribosomal stress, we performed co-IP assays using anti-Ago2 antibodies. As shown in Fig. 6D, treatment of cells with Act D or 5-FU significantly increased the binding of L11 to Ago2. These data demonstrate that L11 recruits Ago2 to c-myc mRNA in response to ribosomal stress. Lastly, we examined whether L11 recruits miR-24 in response to ribosomal stress. As shown in Fig. 6E, treatment of cells with either Act D or 5-FU drastically increased the binding of L11 to miR-24 but not U6 snRNA. In contrast, the total level of miR-24 was not changed following both treatments (Fig. 6F). Together, these data strongly suggest that L11 mediates c-myc mRNA decay by recruiting miR-24-loaded miRISC to c-myc mRNA in response to ribosomal stress.

L11 associates with c-myc mRNA in the cytoplasm.

To test where L11 associates with c-myc mRNA in cells, we performed cell fractionation assays. First, 293 cells transfected with pGL3-myc3′UTR together with control Flag or Flag-L11 vector were then fractionated into cytoplasmic and nuclear fractions (right panel of Fig. 7 A). RNA IP was conducted using the cytoplasmic and nuclear lysates with control IgG or anti-Flag antibodies followed by detection of luciferase and c-myc mRNAs. As shown in Fig. 7A, L11 specifically associates with exogenous luciferase mRNA (left panel) and endogenous c-myc mRNA (middle panel) in the cytoplasm but not in the nucleus. Next, U2OS cells treated with DMSO or Act D were fractionated into cytoplasmic and nuclear lysates (right panel of Fig. 7B) followed by RNA IP using anti-L11 or control IgG. Again, Act D treatment significantly increased the association of endogenous L11 with c-myc mRNA in the cytoplasm (left panel, Fig. 7B). Thus, these results indicate that L11 associates with c-myc mRNA in the cytoplasm and that this association is markedly increased following ribosomal stress.

Ribosome-free L11 associates with c-myc mRNA.

It has been proposed that L11 is released from the nucleolus and intact ribosomes to suppress MDM2 in the nucleoplasm, leading to p53 activation, in response to ribosomal stress (68). To further observe the L11 localization following ribosomal stress, we fractionated U2OS cells treated with control DMSO or Act D into cytoplasmic, nuclear, and nucleolar fractions. As shown in Fig. 7C, the levels of L11 in the nucleolus were reduced whereas its levels in both the nucleus and the cytoplasm increased in response to Act D treatment. To test whether it is the ribosome-free L11 that interacts with c-myc mRNA in the cytoplasm, we performed ribosome fractionation assays using sucrose centrifugation to isolate free RPs (nonribosomal supernatant) from ribosomes and polysomes (42). Nonribosomal supernatant isolated from 293 cells transfected with control or Flag-L11 plasmid was immunoprecipitated with control IgG or anti-Flag antibodies, followed by detection of c-myc mRNA. As shown in Fig. 7D, ribosome-free Flag-L11 specifically associated with c-myc mRNA in cells. Similar experiments were also conducted in U2OS cells treated with DMSO or Act D. As shown in Fig. 7E, Act D treatment significantly increased the association of free endogenous L11 with c-myc mRNA in cells (left panel). Of note, the level of free L11 was increased whereas that of L11 in the polysome pellet was decreased by Act D treatment (right panels of Fig. 7E). Taken together, these results suggest that ribosome-free L11 interacts with c-myc mRNA in cells.

Our RNA IP assays were performed in the presence of EDTA, which disrupts the 80S ribosomes and polysomes (51), suggesting that L11 binding to c-myc mRNA is independent of the translating c-myc mRNA. This notion was further supported by the results showing that L11 associated with the c-myc mRNA regardless of the presence or absence of 20 mM EDTA (Fig. 7F). To further confirm this notion while also testing whether L11 associates with c-myc mRNA through binding to translating c-Myc protein in a ribosome–c-myc mRNA complex, we conducted sequential co-IP followed by RT-qPCR assays. 293 cells transfected with V5–c-Myc in the presence or absence of Flag-L11 were subjected to a first co-IP with anti-Flag antibodies. As shown in Fig. 7G and consistent with our previous reports (12, 17), c-Myc was coimmunoprecipitated with Flag-L11 (lane 4). The Flag-L11-associated protein complex was eluted with Flag peptides. The elution was then subjected to a second co-IP using anti-V5 antibodies. The anti-V5 immunoprecipitates (lane 6, Fig. 7G) specifically represent the c-Myc-associated L11. RNA was extracted from the elution from the first IP with anti-Flag antibody or control IgG as well as the immunoprecipitates from the second IP with anti-V5 antibodies or control IgG, followed by detection of c-myc mRNA by the use of RT-qPCR. As shown in Fig. 7H, c-myc mRNA was detected only in the immunoprecipitates from the first anti-Flag and not in those from the second anti-V5 IP. These results clearly demonstrate that L11 association with c-myc mRNA is independent of its interaction with c-Myc protein (including nascent synthesized c-Myc protein), further supporting the notion that L11 association with c-myc mRNA was not due to ribosome translation of c-myc mRNA. Given all of these observations, we conclude that free L11 interacts with c-myc mRNA in the cytoplasm.

L11 regulation of c-myc mRNA is specific.

To determine whether L11 regulation of c-myc mRNA turnover is a general effect of all individual RPs, we first examined whether knockdown of several other RP genes, including L23, L26, and L29, would also affect c-myc mRNA and protein levels. As shown in Fig. 8 A, unlike knockdown of L11, knockdown of L23, L26, or L29 did not affect the levels of c-myc mRNA (top panel) and protein (middle panel) in U2OS cells. Consistent with those results, overexpression of these RPs did not reduce the levels of c-myc mRNA and protein either (Fig. 8B). Also, neither knockdown nor overexpression of S12 affected the levels of c-myc mRNA and protein (data not shown). Furthermore, RNA IPs were performed using anti-Flag antibodies in cells transfected with Flag-L23 or Flag-L29. As shown in Fig. 8C, unlike L11, neither L23 nor L29 was associated with c-myc mRNA in cells. Taken together, these results suggest that L11 downregulation of c-myc mRNA and protein is a specific rather than a general effect of all individual RPs.

To test whether L11 regulation of c-myc mRNA is also specific to c-myc mRNA rather than a general effect on global mRNA turnover, we examined the levels of a number of other mRNAs upon L11 knockdown. These included the tumor suppressor gene p53, oncogenes mdm2 and mdmx, two labile early-response genes that contain AREs in their 3′-UTR similar to that in c-myc 3′-UTR, c-fos and c-jun (8, 9, 46), and the early-response gene egr1. As shown in Fig. 8D, unlike the results seen with c-myc mRNA, none of these genes were upregulated upon knockdown of L11. Thus, L11 regulation of c-myc mRNA is also not a general effect for all cellular transcripts, although we cannot exclude the possibility that knockdown of L11 might affect the turnover of some other untested mRNAs.