Glucocorticoid receptor interacts with PNRC2 in a ligand-dependent manner to recruit UPF1 for rapid mRNA degradation

Hana Cho1,2, Ok Hyun Park1, Joori Park, Incheol Ryu, Jeonghan Kim, Jesang Ko, and Yoon Ki Kim3

Division of Life Sciences, Korea University, Seoul 136-701, Republic of Korea

Edited by Allan Jacobson, University of Massachusetts Medical School, Worcester, MA, and accepted by the Editorial Board February 11, 2015 (receivedfor review May 23, 2014) Glucocorticoid receptor (GR), which was originally known to function PNRC2, suppressor with morphogenetic effect on genitalia 5 as a nuclear receptor, plays a role in rapid mRNA degradation by (SMG5), SMG6, and SMG7, resulting in rapid mRNA degra- acting as an RNA-binding . The mechanism by which this dation. In SMD, UPF1 is recruited to mRNA via its interaction with process occurs remains unknown. Here, we demonstrate that GR, STAU, which recognizes a stem-loop structure in the 3′UTR or an ′ preloaded onto the 5 UTR of a target mRNA, recruits UPF1 through RNA duplex structure formed by intermolecular mRNA–mRNA proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a or mRNA–long noncoding RNA interactions. The recruited UPF1 ligand-dependent manner, so as to elicit rapid mRNA degradation. interacts with PNRC2 and triggers rapid mRNA degradation We call this process GR-mediated mRNA decay (GMD). Although – GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated (17 19). NMD and SMD are closely related pathways because mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, both require UPF1 and both occur in a translation-dependent we find that GMD is mechanistically distinct from NMD and SMD. manner. HMD, another UPF1-dependent mRNA degradation We also identify de novo cellular GMD substrates using microarray pathway, also requires a translation event, as reviewed else- analysis. Intriguingly, GMD functions in the chemotaxis of human where (20). monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) Here, we demonstrate a novel mRNA decay pathway induced mRNA. Thus, our data provide molecular evidence of a posttran- by ligand-bound GR loaded onto the 5′UTR of target mRNAs. scriptional role of the well-studied nuclear hormone receptor, GR, We call this process GR-mediated mRNA decay (GMD). We which is traditionally considered a transcription factor. show that GMD is a mechanistically unique pathway of UPF1- dependent mRNA decay because it occurs in a translation- glucocorticoid receptor | PNRC2 | UPF1 | glucocorticoid receptor-mediated independent manner. In addition, efficient joining of UPF1 to mRNA decay | Nonsense-mediated mRNA decay GMD machinery requires a glucocorticoid-induced GR–PNRC2 interaction. We also provide evidence that the regulation of t the cellular level, glucocorticoid receptor (GR), which GMD efficiency by a glucocorticoid is necessary for chemotaxis Abelongs to the nuclear receptor superfamily, functions as a transcription factor regulating various physiological processes Significance (1–3). In the presence of a glucocorticoid, which diffuses through theplasmamembraneintothecytoplasm,cytosolicGRbinds to the glucocorticoid. The resulting glucocorticoid–GR com- Glucocorticoid receptor (GR) belongs to the nuclear receptor plex is activated and then enters the nucleus. Once in the nu- superfamily and functions as a transcription factor. GR regu- cleus, GR dimerizes, binds to specific cis-acting elements, and lates various physiological processes, including cell prolif- recruits coregulatory for transcriptional activation or eration, energy homeostasis, and inflammation. In this study, repression (4, 5). we provide molecular evidence for the role of GR in the regu- The majority of the coregulatory proteins commonly contain lation of mRNA stability, which we term GR-mediated mRNA a nuclear receptor box (NR box, also called an LXXLL motif), decay (GMD). Efficient GMD requires a ligand, a GR loaded which is important for interactions between coregulatory pro- onto target mRNA, upstream frameshift 1 (UPF1), and proline- teins and nuclear receptors (4–6). The proline-rich nuclear re- rich nuclear receptor coregulatory protein 2. GMD functions in ceptor coregulatory protein (PNRC), however, is an exception the chemotaxis of human monocytes by targeting chemokine because it interacts with nuclear receptors through an SH3- (C-C motif) ligand 2 mRNA. Thus, we unravel a previously un- binding motif [SD(E)PPSPS] rather than an NR box (7, 8). Two appreciated role of GR, which is traditionally considered a PNRC paralogs, PNRC1 and PNRC2, have been identified in transcription factor, in posttranscriptional regulation. mammalian cells (7, 8). PNRC1 and PNRC2 are believed to play Author contributions: H.C., O.H.P., J.P., I.R., J. Kim, J. Ko, and Y.K.K. designed research; similar roles in nuclear receptor-mediated signaling because they H.C., O.H.P., J.P., I.R., and J. Kim performed research; H.C., O.H.P., J.P., I.R., and Y.K.K. interact with similar groups of nuclear receptors. analyzed data; and H.C., O.H.P., J. Ko, and Y.K.K. wrote the paper. Although PNRC2 is known to function as a coregulatory The authors declare no conflict of interest. protein for nuclear receptors, it has a distinct function in mRNA This article is a PNAS Direct Submission. A.J. is a guest editor invited by the Editorial decay pathways including nonsense-mediated mRNA decay Board. (NMD), staufen (STAU)-mediated mRNA decay (SMD), and Freely available online through the PNAS open access option. – replication-dependent histone mRNA degradation (HMD) (9 Data deposition: The data reported in this paper have been deposited in the Ex- 13). NMD serves as a mechanism of both mRNA quality control pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE49591). and posttranscriptional regulation by selectively recognizing and 1H.C. and O.H.P. contributed equally to this work. degrading cellular transcripts that are abnormal or that contain 2Present address: Department of Biochemistry and Biophysics, School of Medicine and a premature translation termination codon (PTC), as reviewed Dentistry, University of Rochester, Rochester, NY 14642. elsewhere (14–16). A key NMD factor, UPF1, is recruited to 3To whom correspondence should be addressed. Email: [email protected]. a terminating ribosome at a PTC. UPF1 then recruits general This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RNA-degrading enzymes via adaptor/effector proteins such as 1073/pnas.1409612112/-/DCSupplemental.

E1540–E1549 | PNAS | Published online March 16, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1409612112 Downloaded by guest on September 30, 2021 PNAS PLUS 2 of human monocytes; these data are suggestive of the physio- Dex logical importance of GMD. A B siRNA Control IP PNRC PNRC2 α -GR α -GR Results mIgG mIgG GR β-actin PNRC2 Interacts with GR in a Ligand-Dependent Manner, Recruiting UPF1 and Decapping Enzyme 1a to the GR Complex. It has been MYC-PNRC2 reported that GR binds directly to a subset of mRNAs (21, 22). It 19 has also been reported that GR binds to PNRC2 in a ligand- FLAG-UPF1 dependent manner (in a yeast two-hybrid system), albeit under 1 7 DCP1A pcDNA3-FLAG-UPF1 nonphysiological conditions (8, 23). We have shown that PNRC2 C 1 6 physically interacts with UPF1 and decapping enzyme 1a (DCP1A; UPF2 siRNA

a component of the ), thereby triggering NMD Control PNRC2 Control PNRC2 (11). According to these previous reports, we hypothesized that UPF3X GR is complexed with UPF1 and DCP1A via its direct inter- eIF4AIII IP α -GR α -GR mIgG mIgG STAU1 action with PNRC2 in a ligand-dependent manner. GR To test this possibility, we first looked for differences in the β-actin FLAG- UPF1 composition of the GR-containing complex either in the absence 10.2 Before IP After IP or in the presence of dexamethasone (Dex), which is a potent DCP1A synthetic glucocorticoid (24). Immunoprecipitation (IP) experi- RNase A 10.1 ments were performed using α-GR antibody and RNase A-treated β-actin total extracts of HEK293T cells transiently expressing MYC- Before IP PNRC2 and FLAG-UPF1. The cells were either pretreated or RNase A Before IP After IP Dex not treated with Dex before IP. The levels of transiently ex- Dex pressed MYC-PNRC2 and FLAG-UPF1 were comparable to, RT-PCR GAPDH mRNA and lower than, those of endogenous PNRC2 and UPF1, re- spectively (Fig. S1). RT-PCR of endogenous GAPDH mRNA Fig. 1. GR associates with UPF1 and DCP1A via PNRC2. (A) IP experiments α 32 with endogenous GR. HEK293T cells were transiently cotransfected with using -[ P]-dATP and specific oligonucleotides demonstrated plasmids expressing MYC-PNRC2 and FLAG-UPF1. Two days later, the cells efficient removal of cellular RNAs by RNase A treatment before were either treated or not treated with Dex for 3 h. Cell extracts were IPs (Fig. 1A, Lower). The IP results revealed that MYC-PNRC2, prepared and were either not treated or treated with RNase A for 15 min FLAG-UPF1, and endogenous DCP1A coimmunopurified with before IP. The IP experiments were performed using either α-GR antibody or endogenous GR in nine-, seven-, and sixfold greater amounts, a nonspecific mouse IgG (mIgG, control). The protein fractions before and respectively, after Dex treatment (Fig. 1A, Upper). Other NMD after IPs were analyzed using Western blotting with the indicated anti- factors, such as UPF2, UPF3X, and eIF4AIII, and an SMD bodies. The levels of coimmunopurified MYC-PNRC2, FLAG-UPF1, and en- factor, STAU1, were not detectably enriched in GR IP, regard- dogenous DCP1A were normalized to the level of immunopurified GR. The normalized levels obtained from IPs from the extracts of cells not treated less of Dex treatment. These results suggest that Dex treatment with Dex were arbitrarily set to 1.0. For quantitative analysis, threefold serial triggers efficient formation of a GR complex containing PNRC2, dilutions of total cell extracts that were obtained before IP were loaded in UPF1, and DCP1A. the four leftmost lanes (Upper). Efficient digestion of cellular transcripts by Next, we evaluated the role of PNRC2 in the formation of the RNase A treatment was demonstrated by the complete absence of cellular GR-containing complex. To this end, we conducted IP experi- GAPDH mRNA according to RT-PCR using α-[32P]-dATP and specific oligo- ments using α-GR antibody and the extracts of either undepleted nucleotides. For quantitative analysis, total cell RNA samples obtained be- or PNRC2-depleted cells. The cells were pretreated with Dex fore IP were serially diluted twofold and analyzed using RT-PCR. The RT-PCR before IP to stimulate formation of the GR-containing complex. products were loaded in the four leftmost lanes (Lower). (B and C)IP Transfection of PNRC2 siRNA selectively reduced the level of experiments of endogenous GR from the extracts of cells depleted of PNRC2. The cells were transiently transfected with either PNRC2 siRNA or a non- endogenous PNRC2, 2% of the normal level (Fig. 1B). The IP specific control siRNA. Two days later, the cells were retransfected with results showed that after the PNRC2 down-regulation, FLAG- a plasmid expressing FLAG-UPF1. One day later, the cells were treated with UPF1 and endogenous DCP1A coimmunopurified with GR in Dex for 3 h before IP. After that, cell extracts were prepared and treated 5- and 10-fold smaller amounts, respectively (Fig. 1C), suggesting with RNase A before IP. (B) A Western blot of PNRC2 demonstrating specific that PNRC2 stabilizes or serves as a link for the associations down-regulation by siRNA. (C) IP experiments using either the α-GR antibody between GR and UPF1, and between GR and DCP1A. Because or mIgG (control). The levels of coimmunopurified FLAG-UPF1 and endog- GR directly binds to the 5′UTR of specific mRNAs even in the enous DCP1A were normalized to the level of immunopurified GR. The normalized levels obtained from IPs from the extracts of undepleted cells

absence of a ligand (21, 22), it is likely that mRNA-bound GR BIOCHEMISTRY recruits PNRC2 or the PNRC2/UPF1/DCP1A complex to were arbitrarily set to 1.0. All data represent at least two independently performed transfection experiments, IPs, and RT-PCRs. mRNA in the presence of a ligand.

GR-Mediated mRNA Decay Targets a Subset of Cellular mRNAs in least 1.5-fold after Dex treatment (Dataset S1). The GMD a Manner Requiring PNRC2, UPF1, and GR. The results shown in substrates are expected to be down-regulated after Dex treat- Fig. 1 led us to hypothesize that the recruitment of UPF1 and ment. We next compared the list of transcripts down-regulated DCP1A to GR-bound mRNA through PNRC2 may elicit rapid mRNA degradation, which we termed GR-mediated mRNA after Dex treatment with previously reported microarray data on decay. In support of this idea, it was reported that GR binds transcripts coimmunoprecipitated with GR (22), and finally directly to the 5′UTR of chemokine (C-C motif) ligand 2 (CCL2) obtained four natural transcripts that were putative GMD sub- mRNA and CCL7 mRNA, and destabilizes these mRNAs in the strates. Interestingly, among them was CCL2 mRNA, which is presence of Dex (21, 22). To test the above hypothesis, we first known to be rapidly degraded as a result of Dex treatment. The searched for natural GMD substrates by performing microarray others were transcripts encoding B-Cell CLL/lymphoma 3 (BCL3), analysis using 48K human gene chips and total transcripts puri- zinc finger SWIM-type containing 4 (ZSWIM4), and pleckstrin fied from HeLa cells that were either treated or not treated with homology-like domain, family A, member 1 (PHLDA1). Dex. The microarray analysis revealed that 147 and 77 tran- We next tested whether the putative GMD substrates re- scripts were up-regulated and down-regulated, respectively, by at quire PNRC2, UPF1, and GR for efficient decay. To this end,

Cho et al. PNAS | Published online March 16, 2015 | E1541 Downloaded by guest on September 30, 2021 endogenous PNRC2, UPF1, and GR were down-regulated to was almost completely reversed after a down-regulation of GR, 15%, 8%, and 2% of the normal level, respectively, using specific PNRC2, or UPF1 (Fig. 3 B–E). The level of C5′-RLuc-C3′ siRNA (Fig. S2 A and B). After that process, the levels and half- mRNA, which contains both the CCL2 5′UTR and CCL2 lives of all of the putative GMD substrates including CCL7 3′UTR (C3′) upstream and downstream, respectively, of the mRNA, whose expression is known to be reduced by Dex RLuc ORF, was also reduced after Dex treatment and restored treatment (21, 22), were analyzed using quantitative RT-PCR by a down-regulation of PNRC2 or UPF1 (Fig. 3E). On the other (RT-qPCR). The results showed that the levels and half-lives of hand, the level of RLuc-C3′ mRNA, which contains the CCL2 all of the putative GMD substrates were drastically decreased 3′UTR downstream of the RLuc ORF, was not affected by Dex after Dex treatment and were significantly restored via the down- treatment and by down-regulation of PNRC2 or UPF1 (Fig. 3E), regulation of PNRC2, UPF1, or GR (Fig. 2 and Fig. S2 C and suggesting that the 5′UTR of GMD substrates is sufficient to D). In contrast, the level and half-life of endogenous CCL5 trigger GMD. mRNA, which lacks a GR-binding site in its 5′UTR (22), were We further analyzed molecular features of GR binding to the not significantly affected by Dex treatment and by the down- 5′UTR of a GMD substrate. It is known that GR directly binds regulation of PNRC2, UPF1, or GR. All these data indicate that to the region spanning nucleotides 44–60 within the 5′UTR of these transcripts are bona fide substrates of GMD, which requires CCL2 mRNA (22). Deletion of this region from C5′-RLuc PNRC2, UPF1, and GR. mRNA significantly inhibited GMD [C5′(Δ)-RLuc mRNA] (Fig. 3F). We also designed RLuc-C5′ mRNA, which contains the GMD Requires Binding of GR to a Target mRNA in a Position- CCL2 5′UTR downstream, rather than upstream, of the RLuc Independent Manner. To gain molecular insight into GMD, we ORF. Intriguingly, the level of RLuc-C5′ mRNA was signifi- next tested the positional effect of GR binding to mRNA. For cantly reduced after Dex treatment to the extent observed in this purpose, we designed several reporter constructs in which C5′-RLuc mRNA (Fig. 3F). The reduction was reversed by a the 5′UTR, 3′UTR, or both, of CCL2 mRNA, BCL3 mRNA, down-regulation of GR, PNRC2, or UPF1, but not by a down- or PHLDA1 mRNA were inserted immediately upstream or regulation of an NMD-specific factor, UPF2 (Fig. S3). These results downstream of the ORF of Renilla luciferase (RLuc) cDNA (Fig. suggest that loading of GR onto a GR-binding site within target 3A). The levels of reporter mRNAs were monitored using mRNA is sufficient for efficient GMD and that the position of the RT-qPCR. The results showed that Dex treatment reduced the GR-binding site relative to the ORF is not critical for GMD. levels of C5′-RLuc mRNA, B5′-RLuc mRNA, and P5′-RLuc mRNA, which contain the CCL2 5′UTR (C5′), BCL3 5′UTR The Loading of GR onto Target mRNA and Binding of GR to a Ligand (B5′), and PHLDA1 5′UTR (P5′), respectively, upstream of the Are Crucial for GMD. Previous UV cross-linking studies using in RLuc ORF, to ∼40% of untreated levels (Fig. 3 B–E). The reduction vitro-synthesized CCL2 mRNA and the extracts of cells either treated or not treated with Dex revealed that GR directly binds to a GR-binding site within CCL2 mRNA (21, 22). It may be possible, however, that Dex-induced signaling molecules or CCL2 mRNA CCL7 mRNA CCL5 mRNA GR-related proteins other than GR itself are involved in the rec- 100 ognition of a GR-binding site and induce GMD within the cells. To determine whether the loading of GR alone onto a target mRNA 50 is sufficient for GMD, we used an artificial tethering system, the bacteriophage λN-5BoxB system (Fig. 4A). The results showed that artificial tethering of λN-HA-GR, but not of λN-HA-GFP, t = 30.1 t1/2 = 5.7 t1/2 = 7.5 1/2 to the 5′UTR of a reporter mRNA containing five tandem t = 24.6 t1/2 = 4.5 t1/2 = 4.8 1/2 repeats of the bacteriophage BoxB sequence (5BoxB) triggered Percent remaining (%) t = 4.4 t = 4.1 t = 49.5 1/2 1/2 1/2 rapid degradation of the reporter mRNA in the presence of Dex, t1/2 = 2.0 t1/2 = 1.8 t1/2 = 30.1 10 in a way that was completely reversed after down-regulation of 012301230123 PNRC2 and UPF1, but not of UPF2 (Fig. 4B). The Dex-induced Time (h) Time (h) Time (h) rapid degradation of the reporter mRNA elicited by tethered λ BCL3 mRNA ZSWIM4 mRNA PHLDA1 mRNA N-HA-GR was almost completely blocked by either a single 100 amino acid substitution, V729I, which reduces the ligand-binding ability of GR (25), or by deletion of the ligand-binding domain (LBD) of GR (Fig. 4C). A specific down-regulation by siRNA 50 and proper expression of the effectors were demonstrated by Control siRNA, Dex - means of Western blotting (Fig. S4 A–C). These results suggest ′ t = 5.3 t = 4.6 t = 3.5 UPF1 siRNA, Dex + that the binding of GR to the 5 UTR is sufficient for eliciting 1/2 1/2 1/2 GMD and that direct interaction between GR and its ligand is t1/2 = 4.1 t1/2 = 3.4 t1/2 = 2.6 PNRC2 siRNA, Dex +

Percent remaining (%) t = 3.1 t = 3.0 t = 2.2 necessary for GMD. 1/2 1/2 1/2 Control siRNA, Dex + t1/2 = 1.6 t1/2 = 1.3 t1/2 = 1.1 10 An Interaction Between GR and DCP1A via PNRC2 Is Important for 012012 012 Efficient GMD. Because the ligand-dependent interaction between Time (h) Time (h) Time (h) GR and PNRC2 occurs via an SH3-binding motif of PNRC2 Fig. 2. The half-lives of newly identified GMD substrates are increased after (7, 8), we further analyzed the role of the Dex-dependent a down-regulation of PNRC2 or UPF1. HeLa cells were transiently transfected GR–PNRC2 interaction in GMD using a complementation ap- with the indicated siRNAs. Three days after the transfection, the cells were proach. First, we generated siRNA-resistant PNRC2 constructs treated with DRB, which is a potent transcription inhibitor, and then treated [PNRC2(R)] (Fig. S4D) that expressed: (i) MYC-tagged PNRC2 with Dex. After that, total cell RNA and protein were prepared at the in- WT; (ii) a double-mutant P101A/P104A, which has amino acid dicated time points after Dex treatment in the presence of DRB. Specific substitutions from proline to alanine at positions 101 and 104 down-regulation by siRNAs was demonstrated by Western blotting (Fig. S2A). The levels of the mRNAs tested, which were normalized to endogenous located within the SH3-binding motif and renders PNRC2 un- GAPDH mRNA, were plotted as a function of time after Dex treatment. The able to interact with GR (8) without affecting its interactions normalized levels of mRNAs at 0 h were arbitrarily set to 100%. The dots and with UPF1 (10); (iii) W114A; and (iv) P108A, both of which lack bars represent the mean and SD of two independent biological replicates. the DCP1A-binding ability of PNRC2 (12). In this study, we also

E1542 | www.pnas.org/cgi/doi/10.1073/pnas.1409612112 Cho et al. Downloaded by guest on September 30, 2021 A B PNAS PLUS

C

D

EF

Fig. 3. Artificial GMD reporter mRNA harboring a GR-binding site is subject to efficient GMD in a way that depends on GR, PNRC2, and UPF1. (A)A schematic of GMD reporter RLuc constructs used in this study. See Results for details. A plasmid expressing FLuc served as a control of variation in transfection efficiency. C5′, CCL2 5′UTR; C3′, CCL2 3′UTR; B5′, BCL3 5′UTR; P5′, PHLDA1 5′UTR; SL, stem-loop structure; and Δ, a deletion of the GR-binding site. (B–E) The effects of a down-regulation of GR, UPF1, or PNRC2 on GMD reporter RLuc mRNAs. HeLa cells were transfected with the indicated siRNAs. One day later, the cells were transfected with a GMD reporter RLuc plasmid and a FLuc control plasmid. Two days later, the cells were either treated or not treated with Dex for 12 h before harvesting. (B and D) Western blots demonstrating specific down-regulation of GR, UPF1, or PNRC2. (C and E) RT-qPCR analysis of reporter RLuc mRNAs. The levels of GMD reporter RLuc mRNAs were normalized to the levels of FLuc mRNAs. The normalized levels of GMD reporter RLuc mRNAs without Dex treatment were arbitrarily set to 100%. (F) The effect of deletion of a GR-binding site and a positional effect of the GR-binding site. HeLa cells were transfected with the indicated GMD reporter RLuc plasmid and a FLuc control plasmid. Two days later, the cells were either treated or not treated with Dex for 12 h before harvesting. The levels of GMD reporter RLuc mRNAs were normalized as described in Fig. 3C.The columns and bars represent the mean and SD of three independent biological replicates. Two-tailed, equal-sample variance Student′s t tests were used to < calculate the P values. **P 0.01. BIOCHEMISTRY

confirmed that the P101A/P104A mutant failed to interact with the expression level of MYC-PNRC2(R) was too low for a com- endogenous GR even in the presence of Dex, without affecting plementation experiment. Therefore, we used HEK293T instead its interactions with endogenous DCP1A (Fig. S4 E and F). In of HeLa cells to achieve a sufficient level of MYC-PNRC2(R) addition, the W114A and P108A mutants failed to interact with expression that corresponded to the endogenous level of the endogenous DCP1A, without affecting the ability of PNRC2 to PNRC2 protein. interact with FLAG-GR and FLAG-UPF1 (Fig. S4G). Further- Western blotting confirmed that transfection of PNRC2 more,thesemutantsfailedtoassociatewithFLAG-taggedDCP2 siRNA reduced the level of endogenous PNRC2 to 4% of the (Fig. S4H),whichisknowntointeractwith DCP1A, suggesting that level in untransfected cells, and that MYC-PNRC2(R)-WT, PNRC2 indirectly interacts with DCP2 via DCP1A. All these data -P101A/P104A, -W114A, and -P108A were expressed at a level demonstrate the binding specificity of PNRC2 variants. comparable to the endogenous PNRC2 level (Fig. 4D). Under Next, we conducted complementation experiments using the same conditions, the levels of C5′-RLuc reporter mRNA and HEK293T cells, PNRC2 siRNA, and the PNRC2(R) constructs endogenous CCL2 mRNA were analyzed using RT-qPCR (Fig. (Fig. 4 D and E). When HeLa cells were transiently transfected 4E). The results showed that Dex treatment reduced the levels of with an excess amount of a plasmid expressing MYC-PNRC2(R), C5′-RLuc mRNA and endogenous CCL2 mRNA to 44% and

Cho et al. PNAS | Published online March 16, 2015 | E1543 Downloaded by guest on September 30, 2021 A B C ATG TAA Control PNRC2 UPF1 UPF2 RLuc 140 140 RLuc-5 -5BoxB 5Box 120 120 λNB 100 100 mRNA (%)

λN-HA mRNA (%) 80 80 λN GFP 60 * 60 RLuc λN-HA-GFP ** RLuc ** 40 40 λN GR λN-HA-GR(WT) 20 20 HA tag LBD Relative 0 Relative 0 λN GR λN-HA GFP GR GFP GR GR λN-HA-GR(V729I) GR λN-HA V729I Dex GR  LBD GR  LBD λN GR GR V729I GR V729I λN-HA-GR(∆LBD) Dex D E 140 siRNA ** ** ** 120 ** ** Control PNRC2 100 ** **** 80 C5 -RLuc pCMV-MYC- 60 CCL2 40 Mr PNRC2(R) (kDa) 20 W114A P108A WT P101A/P104A 25 0 Relative level of mRNA (%) MYC-PNRC2(R) Dex 15 PNRC2 pCMV-MYC- P101A/ W114A P108A MYC-PNRC2(R) PNRC2(R) P104A β-actin siRNA Control PNRC2

Fig. 4. GMD requires a ligand-binding ability of GR and GR- and DCP1A-binding abilities of PNRC2. (A) A schematic diagram of (i) the tethering reporter construct, RLuc-5′-5BoxB, which contains five tandem repeats of 5BoxB in the 5′UTR, and (ii) the effector construct, which expresses a C-terminally HA-tagged bacteriophage λN polypeptide (λN-HA), λN-HA-GFP, λN-HA-GR, or λN-HA-GR variants. (B) Analysis of rapid mRNA degradation elicited by GR artificially tethered to the 5′UTR. HeLa cells were transiently transfected with the indicated siRNAs and, 1 d later, transfected with a tethering reporter plasmid, an effector plasmid, and pCI-F, which encodes FLuc cDNA and serves as a control for variation in transfection. Two days later, the cells were either treated or not treated with Dex for 30 min before harvesting. Specific down-regulation of PNRC2, UPF1, and UPF2 (Fig. S4A), and comparable expressions of λN-HA-GFP and λN-HA-GR (Fig. S4B) were validated by Western blotting. The levels of RLuc-5′-5BoxB mRNAs were normalized to the levels of FLuc mRNAs. Normalized levels of RLuc-5′-5BoxB mRNAs in the presence of λN-HA were set to 100%. (C) Effects of GR variants on mRNA degradation. Comparable expression of GR variants were determined by Western blotting (Fig. S4C). (D and E) Complementation experiments using siRNA-resistant PNRC2 variants. HEK293T cells were tran- siently transfected with either PNRC2 siRNA or a nonspecific control siRNA. One day after the transfection, the cells were cotransfected with three plasmids: (i) a plasmid expressing siRNA-resistant MYC-PNRC2(R) either WT or its variant (Fig. S4D), (ii) a GMD reporter plasmid expressing C5′-RLuc mRNA, and (iii) a reference plasmid, pCI-F. Two days later, the cells were either treated or not treated with Dex for 12 h before harvesting. (D) Western blotting of endogenous PNRC2 and MYC-PNRC2(R) with either WT or its variants. (E) RT-qPCR analysis of C5′-RLuc mRNA and endogenous CCL2 mRNA. The levels of C5′-RLuc mRNA and endogenous CCL2 mRNA were normalized to the levels of FLuc mRNAs and GAPDH mRNAs, respectively. The normalized levels of C5′-RLuc mRNA and endogenous CCL2 mRNA in the cells not treated with Dex were arbitrarily set to 100%. The columns and bars represent the mean and SD of three independent biological replicates. **P < 0.01, *P < 0.05.

38%, respectively, of untreated levels and that the reductions were not significantly affected by Dex treatment (see SI Results were almost completely reversed by a PNRC2 down-regulation. for more details). Intriguingly, when MYC-PNRC2(R)-WT was expressed, effi- Conversely, we also tested the effect of an NMD-specific cient GMD of both C5′-RLuc mRNA and endogenous CCL2 factor on GMD. To this end, UPF1 (a common factor for GMD mRNA was restored. On the other hand, comparable expression and NMD), GR (a GMD-specific factor), and UPF2 and UPF3X of MYC-PNRC2(R)-P101A/P104A, -W114A, or -P108A failed to (both of which are known NMD factors) were down-regulated restore GMD. These results suggest that the abilities of PNRC2 to using specific siRNA (Fig. 5 A and B). The results of RT-qPCR interact with GR and DCP1A are necessary for efficient GMD. revealed that the levels of all tested endogenous GMD sub- strates, but not of endogenous CCL5 mRNA, which lacks a GMD Is Mechanistically Distinct from NMD and SMD. Because GR-binding site (22), were drastically reduced upon Dex treat- PNRC2 and UPF1 are commonly involved in NMD and SMD ment and restored by a down-regulation of GR or UPF1, but not (10, 11) as well as in GMD (present study), it is possible that of UPF2 or UPF3X (Fig. 5 A and B). there is a cross-talk between NMD/SMD and Dex-mediated It is known that NMD and SMD are coupled to translation signaling or Dex-induced GMD. To first determine whether (17, 26). Given that GMD shares common factors UPF1 and NMD and SMD are affected by Dex treatment, we assessed the PNRC2 with NMD and SMD, it is likely that GMD is also de- changes in expression of NMD and SMD factors and measured pendent on translation. To test this possibility, we designed the efficiencies of NMD and SMD before and after Dex treat- SL-C5′-RLuc and SL-RLuc-C5′ reporter constructs that harbor ment (Fig. S5). The results revealed that the abundances of all a stem-loop (SL) structure with ΔG = −87.8 kcal/mol (Fig. 3A). tested NMD and SMD factors were not significantly changed It is known that an insertion of a stable SL structure (ΔG = −75 after Dex treatment. In addition, NMD and SMD efficiencies kcal/mol) into the 5′UTR blocks ribosome scanning and causes

E1544 | www.pnas.org/cgi/doi/10.1073/pnas.1409612112 Cho et al. Downloaded by guest on September 30, 2021 ACWestern blotting (Fig. S6). The results showed that in IP of GR, PNAS PLUS endogenous GMD substrates (CCL2 mRNA, PHLDA1 mRNA, and BCL3 mRNA) were enriched approximately twofold in a way that was unaffected by Dex treatment (Fig. 6A), indicating that GR is loaded onto GMD substrates independently of a li- gand. On the other hand, lower amounts of the endogenous GMD substrates coimmunopurified with either MYC-PNRC2 or FLAG-UPF1 relative to GR, when the cells were not treated with Dex (Fig. 6A). Dex treatment, however, significantly in- creased the amounts of the coimmunopurified GMD substrates in the IPs of MYC-PNRC2 or FLAG-UPF1 (Fig. 6A). The B D enrichments of GMD substrates in the IPs of FLAG-UPF1 and MYC-PNRC2 were significantly reversed by down-regulation of GR or PNRC2 (Fig. 6B) and by down-regulation of GR or UPF1 (Fig. 6C), respectively. Consistent with a recent report showing that UPF1 promiscuously binds to mRNA (30), the CCL5 mRNA and N-acetyltransferase 9 (NAT9) mRNA, both of which are not targeted for GMD, were also enriched in the IP of FLAG-UPF1, but not in the IP of GR or MYC-PNRC2 (Fig. 6A). However, this enrichment was not affected by Dex treatment. All data suggest that ligand-free GR loaded onto a GR-binding site within mRNA binds to a ligand and recruits PNRC2 and UPF1 to the mRNA.

UPF1 Facilitates the Interaction Between GR and PNRC2. With re- Fig. 5. GMD is mechanistically distinct from NMD. (A and B) Effects of NMD gard to mRNA degradation, the recruitment of PNRC2 to the factors on GMD. HeLa cells were transfected with the indicated siRNAs, and GR-binding site via GR may be sufficient for mRNA degrada- endogenous GMD substrates were analyzed using RT-qPCR. The levels of tion because PNRC2 interacts directly with DCP1A (11) and endogenous GMD substrates were normalized to levels of endogenous stabilizes the interaction between DCP1A and DCP2 (12). If so, GAPDH mRNA. The normalized levels of endogenous GMD substrates in Dex- why does GMD require UPF1? To address this question, we untreated cells were arbitrarily set to 100%. Specific down-regulation by siRNAs was demonstrated by Western blotting (Fig. S5 G and H). (C and D)A performed IPs using extracts of the cells either depleted or not test of translation independence of GMD. HeLa cells were transfected with depleted of UPF1 (Fig. 6D). The cells were pretreated with Dex the indicated GMD reporter RLuc plasmid and the FLuc control plasmid. Two before IP to induce efficient formation of the GR-containing days later, the cells were either treated or not treated with Dex for 12 h complex. The results of IP using α-GR antibody showed that before harvesting. (C) Translation efficiency of GMD reporter RLuc mRNAs. MYC-PNRC2 coimmunopurified with GR in 10-fold smaller The protein activities of RLuc translated from GMD reporter mRNAs were amounts when UPF1 was down-regulated (Fig. 6E). The reciprocal normalized to the levels of the GMD reporter RLuc mRNAs. The normalized IP using MYC-PNRC2 also showed decreased association between ′ ′ activities of RLuc translated from C5 -RLuc mRNA or RLuc-C5 mRNA were GR and MYC-PNRC2 after UPF1 down-regulation (Fig. S6 J and arbitrarily set to 100%. (D) RT-qPCR analysis of GMD reporter RLuc mRNAs. K). All of these results indicate that UPF1 promotes an interaction The levels of RLuc mRNA were normalized to the levels of FLuc mRNAs. The normalized levels of RLuc mRNA in Dex-untreated cells were arbitrarily set to between GR and PNRC2, which leads to more efficient GMD. 100%. The columns and bars represent the mean and SD of three in- dependent biological replicates. **P < 0.01, *P < 0.05. Helicase Activity of UPF1, but Not Phosphorylation of UPF1, Is Required for Efficient GMD. The above findings that UPF1 facili- tates the interaction between GR and PNRC2 led us to examine drastic inhibition of translation (27–29). Consistent with previous which abilities of UPF1 are involved in GMD. First, to test a role studies, translation efficiencies of SL-C5′-RLuc mRNA and of helicase activity of UPF1 in GMD, we performed the com- SL-RLuc-C5′ mRNA were drastically inhibited compared with plementation experiments using UPF1 siRNA and siRNA-resistant translation efficiencies of C5′-RLuc mRNA and RLuc-C5′ mRNA MYC-UPF1-WT or -R843C, which lacks helicase activity (31, 32), (Fig. 5C). Nevertheless, the GMD efficiencies of SL-C5′-RLuc under the modified conditions (Fig. 7 A and B). In Fig. 2, we mRNA and SL-RLuc-C5′ mRNA were comparable to those of observed that the half-lives of endogenous CCL2 and CCL7 C5′-RLuc mRNA and RLuc-C5′ mRNA, respectively (Fig. 5D). mRNAs were increased to almost normal levels upon UPF1 It should be noted that when the same SL structure is inserted down-regulation. On the other hand, steady-state levels of en- BIOCHEMISTRY into the 5′UTR of NMD reporter mRNA, NMD and translation dogenous CCL2 and CCL7 mRNAs were moderately increased of the reporter mRNA are inhibited by ∼3- and 10-fold, re- by UPF1 down-regulation (Fig. 5A). One critical difference spectively (29). These unexpected results suggest that GMD is between the half-life and steady-state experiments was the independent of a translation event. All of the data shown in treatment with a transcription inhibitor 5,6-dichloro-1-β-D-ribo- Fig. 5 indicate that although all three pathways share common furanosylbenzimidazole (DRB) in the half-life experiment. The factors UPF1 and PNRC2, GMD is mechanistically distinct from simple interpretation for the difference in the extent of GMD NMD and SMD. inhibition by UPF1 down-regulation is that Dex may be involved in the transcription of the CCL2 gene, as well as GMD of CCL2 GR Bound to a Target mRNA Recruits PNRC2 to the Target mRNA in mRNA. In support of this idea, down-regulation of UPF1 almost a Ligand-Dependent Manner to Elicit GMD. We next characterized completely inhibited GMDs of artificially designed reporter the molecular hierarchy involved in the recruitment of GMD mRNAs, the transcription of which is driven by the CMV pro- factors to mRNA. We performed IPs using antibody against moter (Figs. 3 C and E and 4B). Therefore, to minimize the a GMD factor and the extracts of cells either depleted or not possible effect of Dex on transcription of CCL2 gene although depleted of GR, PNRC2, or UPF1 (Fig. 6 and Fig. S6). After GMD efficiency is reduced as well, the incubation time with Dex that, the levels of coimmunoprecipitated endogenous GMD was reduced to 1 h in complementation experiments. substrates were analyzed using RT-qPCR. Specific IPs and se- The results of complementation experiments using UPF1 lective down-regulations by siRNAs were demonstrated by siRNA and siRNA-resistant MYC-UPF1 revealed that Dex

Cho et al. PNAS | Published online March 16, 2015 | E1545 Downloaded by guest on September 30, 2021 treatment for 1 h reduced the level of endogenous CCL2 mRNA * ∼ A 6 B 5 * to 50% of the untreated level, and that down-regulation of ** ** UPF1 significantly inhibited the GMD of CCL2 mRNA (Fig. 7 A 5 4 4 and B). Such inhibition was significantly reversed by expression 3 mRNA 3 mRNA of UPF1-WT but not of UPF1-R843C, suggesting that helicase 2 2 activity of UPF1 is involved in efficient GMD. CCL2 CCL2 1 1 Next, we asked if UPF1 phosphorylation contributes to GMD

Relative enrichment of – Relative enrichment of (Fig. 7 C E). In the cases of NMD and SMD, it is well known 0 0 5 * that the phosphorylation of UPF1 by SMG1 kinase is critical for * IP – 4 target mRNA degradation (33 36). Consistent with previous reports, down-regulation of SMG1 significantly inhibited the

mRNA 3 NMDs of endogenous NAT9 and SC35 (1.6 kb) mRNAs. On 2 siRNA Control GR PNRC2 C the other hand, down-regulation of SMG1 did not signifi- PHLDA1 1 5 5 cantly affect the GMDs of endogenous CCL2 and CCL7

Relative enrichment of ** ** 0 4 4 mRNAs, suggesting that GMD occurs independently of UPF1 4 * phosphorylation. In contrast, down-regulation of GR selec- ** 3 3 3 mRNA tively inhibited GMD but not NMD. All these results strengthen 2 2 our conclusion that GMD is mechanistically distinct from NMD CCL2 mRNA 2 1 1 and SMD (Fig. 5). Relative enrichment of

BCL3 1 0 0 GMD Controls Chemotaxis of Monocytes by Regulating CCL2 mRNA

Relative enrichment of IP 0 Stability. It is known that the CCL2 protein belongs to the CC 12 chemokine family and is involved in chemotaxis of various im- 10 mune cells toward a site of injury or infection (37). Because we 8 siRNA Control GR Control UPF1 found that GMD of CCL2 mRNA is dependent on PNRC2, mRNA 6 D UPF1, and GR in this study, we tested whether CCL2-mediated 4 siRNA chemotaxis is influenced by PNRC2, UPF1, and GR. Accord- CCL5 Control 2 UPF1 UPF1 ingly, we first measured the protein levels of secreted CCL2 Relative enrichment of 0 β-actin using an ELISA. Dex treatment reduced the protein levels of 6 secreted CCL2 to ∼50% of the untreated control (Fig. 8B and Fig. 5 siRNA S7B). The reduced protein levels were restored when PNRC2, 4 E Control UPF1 Control UPF1 UPF1, and GR were down-regulated using specific siRNAs to

mRNA 3 IP 16%, 17%, and 9% of normal levels, respectively (Fig. 8A and Fig. 2 α -GR mIgG mIgG α -GR

NAT9 S7A). On the other hand, the reduced protein level of CCL2 was 1 GR not restored by the UPF2 down-regulation (Fig. S7B). Relative enrichment of 0 MYC-PNRC2 We next assessed the effects of the down-regulation of IP 10.1 PNRC2, UPF1, or GR on cell migration in a chemotaxis mi- β-actin crochamber that had two compartments separated by a poly- μ Before IP After IP vinylpyrrolidone-treated membrane with 5- m pores. The lower Dex Dex compartments of wells were filled with a supernatant (medium) from cells that were either treated or not treated with Dex, and Fig. 6. GR bound to mRNA recruits PNRC2 and UPF1 to the target mRNA were either depleted or not depleted of PNRC2, UPF1, or GR. and the GR-PNRC2 association is stabilized by UPF1. (A) RT-qPCR of coim- Human acute monocytic leukemia cell line THP-1 (expressing munoprecipitated endogenous GMD substrates in IPs of endogenous GR, CCL2 receptors on the plasma membrane) was seeded in the MYC-PNRC2, and FLAG-UPF1. HeLa Cells were either transfected or not upper compartments of wells. When THP-1 cells sense CCL2 transfected with plasmid expressing MYC-PNRC2 or FLAG-UPF1. Two days that diffuses from the lower compartment, they are expected to later, the cells were either treated or not treated with Dex for 3 h. IP ex- periments were performed using α-GR, α-MYC, α-FLAG antibody, or a non- migrate toward the lower compartment and to consequently specific mouse IgG (mIgG, control). IP specificity was demonstrated by adhere to the membrane. The number of migrating cells (cells Western blotting (Fig. S6 A–C). The levels of coimmunoprecipitated GMD adherent to the membrane) was counted to determine the substrates relative to GAPDH mRNA were normalized to the levels of the chemotactic index. The results revealed that cell migration was input amount (GMD substrates relative to GAPDH mRNA before IP). The decreased by 50–60% when the lower compartments of wells normalized levels of GMD substrates obtained in IPs using mIgG without Dex contained supernatants from the cells treated with Dex (Fig. treatment were arbitrarily set to 1.0. (B) RT-qPCR of coimmunoprecipitated 8 C and D and Fig. S7 C and D). Intriguingly, the rate of cell endogenous CCL2 mRNA in IPs of FLAG-UPF1. As performed in A, except that migration was restored by a down-regulation of PNRC2, HeLa cells were depleted or not depleted of GR or PNRC2. The cells were UPF1, or GR, but not by UPF2 (Fig. 8 C and D and Fig. S7 C treated with Dex for 3 h before IP. Specific down-regulation (Fig. S6D) and IP specificity (Fig. S6E) were demonstrated by Western blotting. (C) RT-qPCR of and D). These results suggest that GMD plays an important coimmunoprecipitated endogenous CCL2 mRNA in IPs of MYC-PNRC2. As role in the chemotaxis of monocytes via regulation of CCL2 performed in A, except that HeLa cells were depleted or not depleted of GR mRNA stability. or UPF1. The cells were treated with Dex for 3 h before IP. Specific down- regulation (Fig. S6 F and H) and IP specificity (Fig. S6 G and I) were dem- Discussion onstrated by Western blotting. (D and E) IP of endogenous GR using the In this study, we demonstrate a new mRNA decay pathway, extracts of cells depleted or not depleted of UPF1. HeLa cells, either depleted GMD, which is induced by a ligand (glucocorticoid) and requires or not depleted of UPF1, were transiently transfected with the plasmid GR, PNRC2, UPF1, and DCP1A. According to our findings, we expressing MYC-PNRC2. The cells were treated with Dex for 3 h before IP. After that, the cell extracts were treated with RNase A. IPs were performed propose the following molecular details of GMD. GR can bind using α-GR antibody or mIgG. (D) A Western blot demonstrating specific to a GR-binding site of a target mRNA even in the absence of down-regulation of UPF1. (E) IPs using either an antibody against endoge- a ligand (Fig. 8E, Left). In the presence of a ligand (Fig. 8E, nous GR or nonspecific mIgG. The columns and bars represent the mean and Right), on the other hand, the ligand binds to GR, which is SD of two or three independent biological replicates. **P < 0.01; *P < 0.05. preloaded onto a target mRNA, and the resulting ligand-bound

E1546 | www.pnas.org/cgi/doi/10.1073/pnas.1409612112 Cho et al. Downloaded by guest on September 30, 2021 140 UPF2 and SMG1 are not essential for efficient GMD (Figs. 4B, PNAS PLUS * ** ABsiRNA 120 5B, and 7 C–E). Therefore, it is most likely that other cellular ** Control UPF1 100 factors rather than UPF2 may control the helicase ability of pCMV-MYC 80 UPF1 during GMD. In addition, another kinase rather than

-UPF1(R) mRNA (%) 60 SMG1 may act to activate UPF1 in GMD. Indeed, UPF1 is WT R843C 40

MYC-UPF1(R) Relative level of UPF1 CCL2 20 0 β-actin 2 Dex A siRNA B Control PNRC UPF1 pCMV-MYC 160 WT R843C PNRC2 ** -UPF1(R) 140 C UPF1 120 siRNA siRNA Control UPF1 β-actin 100 ** GR Control SMG1 80 60 GR THP-1 chemotaxis C 40 Relative level of CCL2 protein (%) CCL2 protein 20 SMG1 D Dex CCL2 CCL7 CCL5 0 β-actin 140 Dex

120 siRNA E 100

80 Control siRNA 800 D ** 60 160 40 140 600 ** ** ** ** siRNA NAT9 20 ** 120 100 ** ** 400 SC35 0 80 Relative level of mRNA (%) PNRC2 1.6kb Dex 60 200 40 20

siRNA Control GR SMG1 Chemotaxis index (%) 0

siRNA Dex 0 Relative level of mRNA (%) siRNA UPF1 siRNA GR E SMG1 Control

Fig. 7. GMD requires a helicase activity but not phosphorylation of UPF1. (A and B) Complementation experiments using siRNA-resistant UPF1-WT and its R843C variant. HeLa cells were transiently transfected with either UPF1 siRNA or a nonspecific control siRNA. One day after the transfection, the cells were retransfected with a plasmid expressing siRNA-resistant MYC-UPF1(R)-WT or -R843C. Two days later, the cells were either treated or not treated with Dex for 1 h before harvesting. (A) Western blots demonstrating selective down-regulation of UPF1 and comparable levels of exogenously expressed UPF1(R) and endogenous UPF1. (B) RT-qPCR analysis of endogenous CCL2 mRNA. The levels of endogenous CCL2 mRNA were normalized to the levels of endogenous GAPDH mRNAs. The normalized levels of endogenous CCL2 mRNA in the cells not treated with Dex were arbitrarily set to 100%. (C–E) GMD is not dependent on SMG1 activity. HeLa cells were transiently trans- fected with GR siRNA, SMG1 siRNA, or a nonspecific control siRNA. Three days later, the cells were either treated or not treated with Dex for 3 h be- Fig. 8. Chemotaxis of monocytes is mediated by GMD. The lower and upper fore harvesting. (C) Western blots demonstrating selective down-regula- compartments of each well in a chemotaxis microchamber were separated μ tion of GR and SMG1 by siRNAs. (D) RT-qPCR analysis of endogenous CCL2, on a polyvinylpyrrolidone-treated membrane with 5- m pores. The lower CCL7, and CCL5 mRNAs. The levels of endogenous GMD substrates were compartments of wells were filled with the supernatants of HeLa cells de- normalized to the levels of endogenous GAPDH mRNAs. (E) RT-qPCR analysis pleted of either PNRC2 or UPF1 and either not treated or treated with Dex of endogenous NAT9 and SC35 (1.6 kb) mRNAs. The columns and bars rep- for 3 h. The upper compartments were filled with THP-1 cells (a monocyte resent the mean and SD of three independent biological replicates. **P < cell line). After incubation of the microchamber for 3 h at 37 °C, the mem- 0.01; *P < 0.05. branes were removed from the wells. Then, the migrating cells that adhered to the underside of the membranes were stained and counted under a mi- croscope in six randomly selected visual fields in each well. (A) A Western blot of PNRC2 and UPF1, demonstrating specific down-regulation by siRNAs. GR would recruit PNRC2 and eventually DCP1A and UPF1, BIOCHEMISTRY (B) ELISA for the CCL2 protein concentration in the supernatants of the HeLa both of which directly interact with PNRC2 (11). The resulting cells depleted of either PNRC2 or UPF1. The levels of CCL2 protein were complex may be displaced by the scanning ribosome. Nonethe- normalized to the levels of total protein in the supernatants. The normalized less, the complex may quickly reassociate with 5′UTR because of levels of CCL2 without Dex treatment were arbitrarily set to 100%. the intrinsic RNA-binding ability of GR (22). The recruited (C) Analysis of chemotaxis of THP-1 cells. The migrating cells were stained UPF1 may transiently trigger a remodeling of messenger ribo- and images were acquired to demonstrate the effects of either the PNRC2 or μ nucleoprotein particle (mRNP) via its helicase activity (Fig. 7 A UPF1 down-regulation on cell migration. (Scale bars, 20 m.) (D) The che- motactic index. The stained cells were counted under a microscope in six and B), consequently further stabilizing the GR-PNRC2 in- randomly selected visual fields in each well to determine the chemotactic teraction (Fig. 6 D and E and Fig. S6 J and K). Then, the index. The numbers of stained cells without Dex treatment in each visual resulting complex at the GR-binding site would trigger decapp- field were arbitrarily set to 100%. The columns and bars represent the mean ing followed by 5′-to-3′ degradation of the mRNA. The recruited and SD of at least three independent transfection experiments and ELISA or UPF1 may also repress translation initiation before mRNA chemotaxis assay. **P < 0.01. (E) A model of GMD-mediated chemotaxis. In degradation, as observed in NMD (38). the absence of a glucocorticoid, GMD is inactive and its target mRNA (CCL2 In the case of NMD, it has been reported that UPF1 helicase mRNA) is stable and abundant. Consequently, the expressed CCL2 protein – induces chemoattraction of monocytes (Left). In the presence of a glucocor- activity is promoted by UPF2 (39 41) and that SMG1 provides ticoid, GMD is activated, destabilizes CCL2 mRNA, and thereby causes in- a binding platform for UPF1 and UPF2, resulting in the acti- efficient chemoattraction of monocytes (Right). The details are described vation of UPF1 helicase activity (42). However, we observed that in Discussion.

Cho et al. PNAS | Published online March 16, 2015 | E1547 Downloaded by guest on September 30, 2021 known to be phosphorylated by ATR and DNA-PK during HMD crucial for eliciting GMD after Dex treatment. Nonetheless, all (43, 44). GMD substrates tested in this study were shown to contain Recently, genome-wide analysis showed that UPF1 associates a functional GR-binding site in the 5′UTR (Fig. 3). Although with mRNAs throughout the entire sequences and is displaced more GMD substrates should be tested, it seems that there is an from mRNA by an elongating ribosome (30). Therefore, it is evolutionary selective pressure to maintain the GR-binding site possible that the ligand-bound GR-PNRC2 complex bound to in the 5′UTR. This observation points to the possibility of an the GR-binding site of mRNA recruits either a preexisting UPF1 additional regulatory mechanism of through the within close proximity of the GR-binding site or mRNA-free GR-binding site, aside from GMD. A genome-wide survey should UPF1. Our data demonstrate that the latter mechanism is pre- help to uncover a number of additional natural GMD substrates dominant because the amount of coimmunopurified GMD and will identify further molecular details and biological rele- substrate in the IP of UPF1 is significantly increased by Dex treatment (Fig. 6A). The resulting complex elicits efficient vance of GMD. ′ ′ GMD, possibly in the 5 -to-3 direction because of the en- Materials and Methods hancement of decapping by DCP1A. The possible 3′-to-5′ deg- radation of a GMD substrate remains to be tested. Conse- Construction of Plasmids. The details of plasmid construction are provided in SI Materials and Methods. quently, GMD substrates would be down-regulated, affecting various physiological and metabolic pathways, such as chemo- Cell Culture, Transfection, and the siRNA Sequences. The details of cell taxis of monocytes (Fig. 8). culture, transfection, and the siRNA sequences are provided in SI Materials Other groups as well as ours have shown that the C terminus and Methods. of PNRC2 commonly interacts with GR, DCP1A, and UPF1, although the critical residues in the C terminus involved in the The Tethering Assay. HeLa cells were transiently cotransfected with 0.1 μg interactions between PNRC2 and its binding partners are char- of pRL-5′-5BoxB, 0.2 μg of an effector plasmid, and 0.01 μg of pCI-F using acterized (8, 10, 11, 23). Because of the common binding site, Lipofectamine 2000 (Invitrogen). Two days later, the cells were treated with once one or two binding partners interact with PNRC2, the 100 nM of Dex for 30 min. The cells were then harvested, and total cell third binding partner may not be easily recruited to the RNA and protein were purified using TRIzol Reagent (Life Technologies). complex because of steric hindrance. In the present study, however, we showed that PNRC2 is complexed with DCP1A, Quantitative Real-Time RT-PCR and Semiquantitative RT-PCR Using α-[32P]-dATP UPF1, and GR. Therefore, one possible explanation is that and Specific Oligonucleotides. RT-qPCR analyses were performed as described the steric hindrance would be overcome by PNRC2 di- previously (10, 11, 46). The oligonucleotides used in our study are listed in merization, which may provide a sufficient binding platform Dataset S2. for loading of its binding partners. In support of this idea, it has RT-PCR using α-[32P]-dATP and specific oligonucleotides was performed as recently been reported that PNRC1, an isoform of PNRC2, described previously (11, 17). The 32P-labeled PCR products were analyzed forms a dimer (45). using 5% polyacrylamide gel electrophoresis, visualized with Phosphor- It is generally considered that cytosolic GR enters the nucleus Imaging (BAS-2500; Fuji Photo Film), and then quantitated using Multi- when it binds to a ligand. However, we observed that although Gauge (Fuji Photo Film). most of the cytosolic GR moved to the nuclear fraction upon Dex treatment, a significant amount of GR remained in the cy- Immunoprecipitation. HeLa cells and HEK293T cells were transiently trans- toplasmic fractions (Fig. S8). Moreover, the intracellular distri- fected with the indicated plasmids using Lipofectamine 2000 and the calcium phosphate precipitation method, respectively. The IPs and RNA IPs were bution of PNRC2 was only marginally affected by Dex treatment. performed as described previously (9, 11, 46). Therefore, the GR and PNRC2 remaining in the cytoplasm upon Dex treatment might be sufficient for eliciting GMD. Western Blotting. Antibodies against the following proteins or peptides were Here, we also demonstrate that GMD is mechanistically dis- β — used in this study: FLAG and -actin (Sigma-Aldrich); MYC (Calbiochem), UPF1 tinct from other UPF1-dependent mRNA decay pathways (a gift from Lynne E. Maquat, University of Rochester, Rochester, NY), PNRC2 — NMD, SMD, and HMD although all these pathways share (11), and human STAU1 (47); DCP1A, UPF2, eIF4AIII, phospho-S1078-UPF1, some common factors: UPF1, PNRC2, and DCP1A. One major and phospho-S1096-UPF1 (10); UPF3X (48), eIF4E (Cell Signaling Technology), difference is the method of recruitment of UPF1 to the working CBP80 (49), CBP80/20-dependent translation initiation factor (CTIF; ref. 46), site on mRNA and the method of stabilization of the UPF1- GR (BD Biosciences and Santa Cruz Biotechnology), snRNP70 (Santa Cruz), containing complex. In the case of NMD, either mRNA-unbound and SMG1 (Bethyl). The antibody against UPF1 phosphorylated at threonine or prebound UPF1 is recruited to a terminating ribosome and 28 was raised in rabbits using the synthetic peptide LGADpTQGSEF (AbClon). is stabilized by an downstream of PTC. In the case of SMD and HMD, UPF1 is also recruited to a Microarray Analysis. Microarray analysis was performed by Macrogen, as terminating ribosome during translation and is stabilized by described previously (9, 10, 50). The microarray data were deposited in the downstream STAU and SL binding protein, respectively. Alter- National Center for Biotechnology Information Gene Expression Omnibus natively, UPF1 could be recruited to mRNA via direct in- web-based data repository (series ID: GSE49591). teraction with STAU or SL binding protein and then could join the termination complex. Therefore, NMD, SMD, and HMD are CCL2 Measurements and Chemotaxis Assay. The details of CCL2 measurements tightly coupled to translation events, although the exact timing of and the chemotaxis assay are provided in SI Materials and Methods. UPF1 recruitment is not clear at this point. In the case of GMD, Statistical Analysis. Two-tailed, equal-sample variance Student’s t-tests were however, UPF1 recruitment to the working site on mRNA occurs < independently of translation (Fig. 5 C and D). Instead, the UPF1 used to calculate the P-values. Differences with P 0.05 were considered statistically significant. recruitment is mediated by its direct interaction with PNRC2, which bridges mRNA-bound GR and UPF1 in a Dex-dependent ACKNOWLEDGMENTS. We thank Dr. Lynne E. Maquat for providing nonsense- manner. In addition, GMD is not affected by down-regulation mediated mRNA decay reporter plasmids and the α-UPF1 antibody; Jens Lykke- of known NMD-specific factors (Fig. 5). Therefore, GMD Andersen for pcDNA3-FLAG-DCP2; and Juan Ortín for α-human STAU1 antibody. is a previously unidentified translation-independent and UPF1- This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIP) (2012R1A2A1A01002469 and dependent mRNA decay pathway. 2014R1A2A1A11050412); O.H.P. was in part supported by the Global PhD Another novel feature of GMD is that the presence, rather Fellowship Program through the National Research Foundation funded by than a relative position, of the GR-binding site within mRNA is the Korean Government.

E1548 | www.pnas.org/cgi/doi/10.1073/pnas.1409612112 Cho et al. Downloaded by guest on September 30, 2021 1. Vandevyver S, Dejager L, Libert C (2012) On the trail of the glucocorticoid receptor: 27. Kozak M (1986) Influences of mRNA secondary structure on initiation by eukaryotic PNAS PLUS Into the nucleus and back. Traffic 13(3):364–374. ribosomes. Proc Natl Acad Sci USA 83(9):2850–2854. 2. Oakley RH, Cidlowski JA (2011) Cellular processing of the glucocorticoid receptor 28. Vassilenko KS, Alekhina OM, Dmitriev SE, Shatsky IN, Spirin AS (2011) Unidirectional gene and protein: new mechanisms for generating tissue-specific actions of gluco- constant rate motion of the ribosomal scanning particle during eukaryotic translation corticoids. J Biol Chem 286(5):3177–3184. initiation. Nucleic Acids Res 39(13):5555–5567. 3. Santos GM, Fairall L, Schwabe JW (2011) Negative regulation by nuclear receptors: A 29. Choe J, et al. (2014) eIF4AIII enhances translation of nuclear cap-binding complex- – plethora of mechanisms. Trends Endocrinol Metab 22(3):87 93. bound mRNAs by promoting disruption of secondary structures in 5’UTR. Proc Natl ’ 4. Lonard DM, O Malley BW (2012) Nuclear receptor coregulators: Modulators of pa- Acad Sci USA 111(43):E4577–E4586. – thology and therapeutic targets. Nat Rev Endocrinol 8(10):598 604. 30. Zünd D, Gruber AR, Zavolan M, Mühlemann O (2013) Translation-dependent dis- 5. Kato S, Yokoyama A, Fujiki R (2011) Nuclear receptor coregulators merge transcrip- placement of UPF1 from coding sequences causes its enrichment in 3′ UTRs. Nat Struct – tional coregulation with epigenetic regulation. Trends Biochem Sci 36(5):272 281. Mol Biol 20(8):936–943. 6. Heery DM, Kalkhoven E, Hoare S, Parker MG (1997) A signature motif in transcrip- 31. Sun X, Perlick HA, Dietz HC, Maquat LE (1998) A mutated human homologue to yeast tional co-activators mediates binding to nuclear receptors. Nature 387(6634):733–736. Upf1 protein has a dominant-negative effect on the decay of nonsense-containing 7. Zhou D, et al. (2000) PNRC: A proline-rich nuclear receptor coregulatory protein that mRNAs in mammalian cells. Proc Natl Acad Sci USA 95(17):10009–10014. modulates transcriptional activation of multiple nuclear receptors including orphan 32. Kurosaki T, et al. (2014) A post-translational regulatory switch on UPF1 controls tar- receptors SF1 (steroidogenic factor 1) and ERRalpha1 (estrogen related receptor geted mRNA degradation. Dev 28(17):1900–1916. alpha-1). Mol Endocrinol 14(7):986–998. 33. Ohnishi T, et al. (2003) Phosphorylation of hUPF1 induces formation of mRNA sur- 8. Zhou D, Chen S (2001) PNRC2 is a 16 kDa that interacts with nuclear re- veillance complexes containing hSMG-5 and hSMG-7. Mol Cell 12(5):1187–1200. ceptors through an SH3-binding motif. Nucleic Acids Res 29(19):3939–3948. 34. Yamashita A, Kashima I, Ohno S (2005) The role of SMG-1 in nonsense-mediated 9. Cho H, et al. (2013) SMG5-PNRC2 is functionally dominant compared with SMG5- – SMG7 in mammalian nonsense-mediated mRNA decay. Nucleic Acids Res 41(2): mRNA decay. Biochim Biophys Acta 1754(1-2):305 315. 1319–1328. 35. Yamashita A, Ohnishi T, Kashima I, Taya Y, Ohno S (2001) Human SMG-1, a novel 10. Cho H, et al. (2012) Staufen1-mediated mRNA decay functions in adipogenesis. Mol phosphatidylinositol 3-kinase-related protein kinase, associates with components of Cell 46(4):495–506. the mRNA surveillance complex and is involved in the regulation of nonsense-medi- 11. Cho H, Kim KM, Kim YK (2009) Human proline-rich nuclear receptor coregulatory ated mRNA decay. Genes Dev 15(17):2215–2228. protein 2 mediates an interaction between mRNA surveillance machinery and de- 36. Cho H, Han S, Park OH, Kim YK (2013) SMG1 regulates adipogenesis via targeting of capping complex. Mol Cell 33(1):75–86. staufen1-mediated mRNA decay. Biochim Biophys Acta 1829(12):1276–1287. 12. Lai T, et al. (2012) Structural basis of the PNRC2-mediated link between mrna sur- 37. Myers SJ, Wong LM, Charo IF (1995) Signal transduction and ligand specificity of the veillance and decapping. Structure 20(12):2025–2037. human monocyte chemoattractant protein-1 receptor in transfected embryonic kid- 13. Choe J, Ahn SH, Kim YK (2014) The mRNP remodeling mediated by UPF1 promotes ney cells. J Biol Chem 270(11):5786–5792. rapid degradation of replication-dependent histone mRNA. Nucleic Acids Res 42(14): 38. Isken O, et al. (2008) Upf1 phosphorylation triggers translational repression during 9334–9349. nonsense-mediated mRNA decay. Cell 133(2):314–327. 14. Karam R, Wengrod J, Gardner LB, Wilkinson MF (2013) Regulation of nonsense- 39. Clerici M, et al. (2009) Unusual bipartite mode of interaction between the nonsense- mediated mRNA decay: implications for physiology and disease. Biochim Biophys Acta mediated decay factors, UPF1 and UPF2. EMBO J 28(15):2293–2306. 1829(6-7):624–633. 40. Chamieh H, Ballut L, Bonneau F, Le Hir H (2008) NMD factors UPF2 and UPF3 bridge 15. Schoenberg DR, Maquat LE (2012) Regulation of cytoplasmic mRNA decay. Nat Rev UPF1 to the exon junction complex and stimulate its RNA helicase activity. Nat Struct Genet 13(4):246–259. Mol Biol 15(1):85–93. 16. Schweingruber C, Rufener SC, Zünd D, Yamashita A, Mühlemann O (2013) Nonsense- 41. Chakrabarti S, et al. (2011) Molecular mechanisms for the RNA-dependent ATPase — mediated mRNA decay Mechanisms of substrate mRNA recognition and degrada- activity of Upf1 and its regulation by Upf2. Mol Cell 41(6):693–703. – tion in mammalian cells. Biochim Biophys Acta 1829(6-7):612 623. 42. Melero R, et al. (2014) Structures of SMG1-UPFs complexes: SMG1 contributes to 17. Kim YK, Furic L, Desgroseillers L, Maquat LE (2005) Mammalian Staufen1 recruits Upf1 regulate UPF2-dependent activation of UPF1 in NMD. Structure 22(8):1105–1119. ’ – to specific mRNA 3 UTRs so as to elicit mRNA decay. Cell 120(2):195 208. 43. Kaygun H, Marzluff WF (2005) Regulated degradation of replication-dependent 18. Park E, Maquat LE (2013) Staufen-mediated mRNA decay. Wiley Interdiscip Rev RNA histone mRNAs requires both ATR and Upf1. Nat Struct Mol Biol 12(9):794–800. – 4(4):423 435. 44. Müller B, Blackburn J, Feijoo C, Zhao X, Smythe C (2007) DNA-activated protein kinase 19. Gong C, Maquat LE (2011) lncRNAs transactivate STAU1-mediated mRNA decay by functions in a newly observed S phase checkpoint that links histone mRNA abundance duplexing with 3′ UTRs via Alu elements. Nature 470(7333):284–288. with DNA replication. J Cell Biol 179(7):1385–1398. 20. Marzluff WF, Wagner EJ, Duronio RJ (2008) Metabolism and regulation of canonical 45. Zhou D, et al. (2007) PNRC is a unique nuclear receptor coactivator that stimulates histone mRNAs: Life without a poly(A) tail. Nat Rev Genet 9(11):843–854. RNA polymerase III-dependent transcription. J Mol Signal 2:5. 21. Dhawan L, Liu B, Blaxall BC, Taubman MB (2007) A novel role for the glucocorticoid 46. Kim KM, et al. (2009) A new MIF4G domain-containing protein, CTIF, directs nuclear receptor in the regulation of monocyte chemoattractant protein-1 mRNA stability. cap-binding protein CBP80/20-dependent translation. Genes Dev 23(17):2033–2045. J Biol Chem 282(14):10146–10152. 47. Marión RM, Fortes P, Beloso A, Dotti C, Ortín J (1999) A human sequence homologue 22. Ishmael FT, et al. (2011) The human glucocorticoid receptor as an RNA-binding pro- of Staufen is an RNA-binding protein that is associated with polysomes and localizes tein: Global analysis of glucocorticoid receptor-associated transcripts and identifica- – tion of a target RNA motif. J Immunol 186(2):1189–1198. to the rough endoplasmic reticulum. Mol Cell Biol 19(3):2212 2219. 23. Zhou D, Ye JJ, Li Y, Lui K, Chen S (2006) The molecular basis of the interaction be- 48. Kim KM, Cho H, Kim YK (2012) The upstream open reading frame of cyclin-dependent tween the proline-rich SH3-binding motif of PNRC and estrogen receptor alpha. kinase inhibitor 1A mRNA negatively regulates translation of the downstream main – Nucleic Acids Res 34(20):5974–5986. open reading frame. Biochem Biophys Res Commun 424(3):469 475. 24. Schäcke H, Döcke WD, Asadullah K (2002) Mechanisms involved in the side effects of 49. Choe J, et al. (2012) Translation initiation on mRNAs bound by nuclear cap-binding glucocorticoids. Pharmacol Ther 96(1):23–43. protein complex CBP80/20 requires interaction between CBP80/20-dependent trans- 25. Malchoff DM, et al. (1993) A mutation of the glucocorticoid receptor in primary lation initiation factor and eukaryotic translation initiation factor 3g. J Biol Chem cortisol resistance. J Clin Invest 91(5):1918–1925. 287(22):18500–18509. 26. Ishigaki Y, Li X, Serin G, Maquat LE (2001) Evidence for a pioneer round of mRNA 50. Cho H, Ahn SH, Kim KM, Kim YK (2013) Non-structural protein 1 of influenza viruses translation: mRNAs subject to nonsense-mediated decay in mammalian cells are inhibits rapid mRNA degradation mediated by double-stranded RNA-binding protein, – – bound by CBP80 and CBP20. Cell 106(5):607 617. staufen1. FEBS Lett 587(14):2118 2124. BIOCHEMISTRY

Cho et al. PNAS | Published online March 16, 2015 | E1549 Downloaded by guest on September 30, 2021