Nonsense-mediated mRNA decay (NMD) is the best-characterized mRNA surveillance mechanism by which aberrant mRNAs harboring premature termination codons are degraded before translation. However, to date, how NMD machinery recruits the general decay complex to faulty mRNAs and degrades those mRNAs remains unclear. Here we identify human proline-rich nuclear receptor coregulatory protein 2 (PNRC2) as a Upf1- and Dcp1a-interacting protein. Downregulation of PNRC2 abrogates NMD, and artificially tethering PNRC2 downstream of a normal termination codon reduces mRNA abundance. Accordingly, PNRC2 preferentially interacts with hyperphosphorylated Upf1 compared with wild-type Upf1 and triggers movement of hyperphosphorylated Upf1 into processing bodies (P bodies). Our observations suggest that PNRC2 plays an essential role in mammalian NMD, mediating the interaction between the NMD machinery and the decapping complex, so as to target the aberrant mRNA-containing RNPs into P bodies.

and has received compensation from these companies in the form of stock; A.R.K. is a research collaborator of Ionis Pharmaceuticals and has received royalty income from Ionis through his employer, Cold Spring Harbor Laboratory. O.A.-W. has served as a consultant for H3 Biomedicine, Foundation Medicine Inc., Merck, and Janssen; O.A.-W. has received personal speaking fees from Daiichi Sankyo. O.A.-W. has received prior research funding from H3 Biomedicine unrelated to the current manuscript. D.I., R.K.B. and O.A.-W. are inventors on a provisional patent application (patent number FHCC.P0044US.P) applied for by Fred Hutchinson Cancer Research Center on the role of reactivating BRD9 expression in cancer by modulating aberrant BRD9 splicing in SF3B1 mutant cells.

Nonsense-mediated mRNA decay (NMD) is an important mRNA surveillance system, and human PNRC2 protein mediates the link between mRNA surveillance and decapping. However, the mechanism by which PNRC2 interacts with the mRNA surveillance machinery and stimulates NMD is unknown. Here, we present the crystal structure of Dcp1a in complex with PNRC2. The proline-rich region of PNRC2 is bound to the EVH1 domain of Dcp1a, while its NR-box mediates the interaction with the hyperphosphorylated Upf1. The mode of PNRC2 interaction with Dcp1a is distinct from those observed in other EVH1/proline-rich ligands interactions. Disruption of the interaction of PNRC2 with Dcp1a abolishes its P-body localization and ability to promote mRNA degradation when tethered to mRNAs. PNRC2 acts in synergy with Dcp1a to stimulate the decapping activity of Dcp2 by bridging the interaction between Dcp1a and Dcp2, suggesting that PNRC2 is a decapping coactivator in addition to its adaptor role in NMD.

Glucocorticoid receptor (GR), which was originally known to function as a nuclear receptor, plays a role in rapid mRNA degradation by acting as an RNA-binding protein. The mechanism by which this process occurs remains unknown. Here, we demonstrate that GR, preloaded onto the 5′UTR of a target mRNA, recruits UPF1 through proline-rich nuclear receptor coregulatory protein 2 (PNRC2) in a ligand-dependent manner, so as to elicit rapid mRNA degradation. We call this process GR-mediated mRNA decay (GMD). Although GMD, nonsense-mediated mRNA decay (NMD), and staufen-mediated mRNA decay (SMD) share upstream frameshift 1 (UPF1) and PNRC2, we find that GMD is mechanistically distinct from NMD and SMD. We also identify de novo cellular GMD substrates using microarray analysis. Intriguingly, GMD functions in the chemotaxis of human monocytes by targeting chemokine (C-C motif) ligand 2 (CCL2) mRNA. Thus, our data provide molecular evidence of a posttranscriptional role of the well-studied nuclear hormone receptor, GR, which is traditionally considered a transcription factor. glucocorticoid receptor | PNRC2 | UPF1 | glucocorticoid receptor-mediated mRNA decay | Nonsense-mediated mRNA decay A t the cellular level, glucocorticoid receptor (GR), which belongs to the nuclear receptor superfamily, functions as a transcription factor regulating various physiological processes (1-3). In the presence of a glucocorticoid, which diffuses through the plasma membrane into the cytoplasm, cytosolic GR binds to the glucocorticoid. The resulting glucocorticoid-GR complex is activated and then enters the nucleus. Once in the nucleus, GR dimerizes, binds to specific cis-acting elements, and recruits coregulatory proteins for transcriptional activation or repression (4, 5).The majority of the coregulatory proteins commonly contain a nuclear receptor box (NR box, also called an LXXLL motif), which is important for interactions between coregulatory proteins and nuclear receptors (4-6). The proline-rich nuclear receptor coregulatory protein (PNRC), however, is an exception because it interacts with nuclear receptors through an SH3-binding motif [SD(E)PPSPS] rather than an NR box (7,8). Two PNRC paralogs, PNRC1 and PNRC2, have been identified in mammalian cells (7,8). PNRC1 and PNRC2 are believed to play similar roles in nuclear receptor-mediated signaling because they interact with similar groups of nuclear receptors.Although PNRC2 is known to function as a coregulatory protein for nuclear receptors, it has a distinct function in mRNA decay pathways including nonsense-mediated mRNA decay (NMD), staufen (STAU)-mediated mRNA decay (SMD), and replication-dependent histone mRNA degradation (HMD) (9-13). NMD serves as a mechanism of both mRNA quality control and posttranscriptional regulation by selectively recognizing and degrading cellular transcripts that are abnormal or that contain a premature translation termination codon (PTC), as reviewed elsewhere (14-16). A key NMD factor, UPF1, is recruited to a terminating ribosome at a PTC. UPF1 then recr...

We previously demonstrated that the receptor for the complement component C1q (gC1qR) is a lipid raft protein that is indispensable for adipogenesis and insulin signaling. Here, we provide the first report that gC1qR is an essential component of lamellipodia in human lung carcinoma A549 cells. Cell-surface gC1qR was concentrated in the lamellipodia along with CD44, monosialoganglioside, actin, and phosphorylated focal adhesion kinase in cells stimulated with insulin, IGF-1, EGF, or serum. The growth factor-induced lamellipodia formation and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases. Moreover, the gC1qR-depleted cells exhibited a reduced proliferation rate in culture as well as diminished tumorigenic and metastatic activities in grafted mice. We therefore conclude that cell-surface gC1qR regulates lamellipodia formation and metastasis via receptor tyrosine kinase activation.

Phosphatidylionsitol 4,5-bisphosphate (PIP2), a substrate of phospholipase C, has recently been recognized to regulate membraneassociated proteins and act as a signal molecule in phospholipase C-linked Gq-coupled receptor (GqPCR) pathways. However, it is not known whether PIP 2 depletion induced by GqPCRs can act as receptor-specific signals in native cells. We investigated this issue in cardiomyocytes where PIP2-dependent ion channels, G proteingated inwardly rectifying K ؉ (GIRK) and inwardly rectifying background K ؉ (IRK) channels, and various GqPCRs are present. The GIRK current was recorded by using the patch-clamp technique during the application of 10 M acetylcholine. The extent of receptor-mediated inhibition was estimated as the current decrease over 4 min while taking the GIRK current (I GIRK) value during a previous stimulation as the control. Each GqPCR agonist inhibited IGIRK with different potencies and kinetics. The extents of inhibition induced by phenylephrine, angiotensin II, endothelin-1, prostaglandin F2␣, and bradykinin at supramaximal concentrations were (mean ؎ SE) 32.1 ؎ 0.6%, 21.9 ؎ 1.4%, 86.4 ؎ 1.6%, 63.7 ؎ 4.9%, and 5.7 ؎ 1.9%, respectively. GqPCR-induced inhibitions of IGIRK were not affected by protein kinase C inhibitor (calphostin C) but potentiated and became irreversible when the replenishment of PIP2 was blocked by wortmannin (phosphatidylinositol kinase inhibitor). Loading the cells with PIP2 significantly reduced endothelin-1 and prostaglandin F2␣-induced inhibition of IGIRK. On the contrary, GqPCR-mediated inhibitions of inwardly rectifying background K ؉ currents were observed only when GqPCR agonists were applied with wortmannin, and the effects were not parallel with those on IGIRK. These results indicate that GqPCR-induced inhibition of ion channels by means of PIP2 depletion occurs in a receptor-specific manner.phospholipase C ͉ cardiac myocyte ͉ phosphatidylinositol 4-kinase ͉ Gq-coupled receptor ͉ G protein-gated inwardly rectifying K ϩ channels

We have shown previously that cardiac G protein-gated inwardly rectifying K ؉ (GIRK) channels are inhibited by Gq protein-coupled receptors (GqPCRs) via phosphatidylinositol 4,5-bisphosphate (PIP2) depletion in a receptor-specific manner. To investigate the mechanism of receptor specificity, we examined whether the activation of GqPCRs induces localized PIP2 depletion. When we applied endothelin-1 to the bath, GIRK channel activities recorded in cell-attached patches were not changed, implying that PIP2 signal is not diffusible but is a localized signal. To test this possibility, we directly measured lateral diffusion by introducing fluorescence-labeled phosphoinositides to a small area of the membrane with patch pipettes. After pipettes were attached, phosphatidylinositol 4-monophosphate or phosphatidylinositol diffused rapidly to the entire membrane, whereas PIP2 was confined to the membrane patch inside the pipette. The confinement of PIP2 was disrupted after cytochalasin D treatment, suggesting that the cytoskeleton is responsible for the low mobility of PIP2. The diffusion coefficient (D) of PIP2 in the plasma membrane measured with the fluorescence recovery after photobleaching technique was 0.00039 m 2 ͞s (n ‫؍‬ 6), which is markedly lower than D of phosphatidylinositol (5.8 m 2 ͞s, n ‫؍‬ 5). Simulation of PIP2 concentration profiles by the diffusion model confirms that when D is small, the kinetics of PIP2 depletion at different distances from phospholipase C becomes similar to the characteristic kinetics of GIRK inhibition by different agonists. These results imply that PIP2 depletion is localized adjacent to GqPCRs because of its low mobility, and that spatial proximity of GqPCR and the target protein underlies the receptor specificity of PIP2-mediated signaling. P rotein-lipid interactions have been increasingly appreciated in recent years. In particular, phosphatidylinositol 4,5-bisphosphate (PIP 2 ) binds a wide variety of cellular proteins, including cytoskeletal proteins and ion channels, and evidence that the binding is essential for their functions is accumulating (1, 2). Because many proteins are known to be regulated by PIP 2 , the question arises as to how a particular protein, among many others, is selectively regulated by PIP 2 changes generated by specific signals. In fact, the same question has been asked for many years in the context of the signaling role of Ca 2ϩ , i.e., ''how are so many different Ca 2ϩ -dependent reactions selectively choreographed within a cell?'' The results of numerous studies undertaken to answer this question have led to the idea that the secret lies in localization, i.e., cells have many means of generating intracellular Ca 2ϩ signals, and the spatial proximities of these signals to the molecular targets of Ca 2ϩ determine the specificity of Ca 2ϩ action (3). However, it is not known whether this idea can be applied to PIP 2 -dependent signaling.Because experimental methods of measuring or visualizing changes in PIP 2 during signaling pathway stimulation remain ...

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