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 protein. 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 proteins 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 Gene 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 -GR -GR PNRC2 α α 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 decapping complex), thereby triggering NMD Control PNRC2 Control PNRC2 (11). According to these previous reports, we hypothesized that UPF3X eIF4AIII IP -GR GR is complexed with UPF1 and DCP1A via its direct inter- -GR α α 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.