Rasd1 Modulates the Coactivator Function of NonO Figure 4. Rasd1 requires full-length NonO to suppress the cAMP pathway in HEK293T cells. (A) Schematic drawing of the locations of specific domains of NonO protein and its truncated constructs. Q, glutamine-rich region; RRM, RNA-recognition motif; HTH, helix-turn-helix and highly-charged region; P, proline-rich region. Bipartite NLS is located within HTH. (B) HEK293T cells were transfected with pGST-Rasd1 along with various constructs of NonO-V5. Lysates were then incubated with MagneGSTTM particles, which enable binding of GST-Rasd1. Only NonOD2-V5 did not interact with GST-Rasd1 (Lane 4). Anti-V5 and Anti-Xpress were used for detection of LacZ and all truncated clones of NonO; anti-GST was used for detection of GST-Rasd1. (C) Rasd1 (2 mg) was co-transfected with either NonO or NonO mutants (2 mg) and luciferase assay was performed subsequently. Neither mutant was able to repress CREB’s activity in the presence of Rasd1 unlike that of wild-type NonO (compare Histograms II with III and IV). (D) Immunofluorescence studies of NonOD1 and Rasd1 in HEK293T cells. GST-NonOD1 is primarily localised in the cytoplasm (Figure D2). In the event of co-transfection with GST-NonOD1, His-Rasd1 is localised in the cytoplasm, whereas the sub-cellular distribution of His-Rasd1 was concentrated in the nucleus in the presence of NonO (compare Figure 3 A8 with Figure 4 D5). doi:10.1371/journal.pone.0024401.g004 different mechanisms. These include sequestration of the co- activator, TORC, that is normally anchored in the cytoplasm by 14-3-3 proteins in the absence of stimulation of the pathway [7,8]; and competition for limiting factor such as CBP, which is a co- activator required for multiple signaling pathways [9]. In this study, our findings lend credence to a new mode of regulation of co-activators of the cAMP pathway – modulation of co-activator function. We define modulation in the context whereby the identity of an interacting partner of the co-activator determines the role of the co-activator in regulation of the target gene upon induction of the cAMP pathway. NR4A1 and NR4A2 are clock-controlled genes oscillating in multiple tissues [53,54] and are CREB-target genes whose expressions are up-regulated upon activation of the cAMP pathway [13]. The nuclear orphan receptors 4A (NR4A) subgroup belongs to the nuclear hormone receptor family and consists of transcription factors capable of recognising the NGFI-B response element (NBRE) [55,56,57,58]. NR4A proteins can bind to DNA as monomers, homodimers and heterodimers [59,60]. Transcrip- tion factor NR4A2 is an immediate early gene induced by many external stimuli, including retinoic acid, forskolin, prostaglandin E2, and dexamethasone [51,56,61,62]. NR4A proteins play important roles in metabolism and in the pathogenesis of various diseases including colorectal cancer, Alzheimer’s disease, familial Parkinson’s disease, schizophrenia, inflammatory arthritis, and manic depression [51,56,61,63,64,65,66]. In addition, NR4A proteins also play a crucial part in CREB-dependent neuro- protection, cell survival and cell transformation of HeLa cells [52,63,67,68]. Expression of NR4A proteins is up-regulated by the cAMP pathway for initiation of the survival of HeLa cancer cells [68]. NR4A proteins are activated via the cAMP pathway through PGE2 in human colorectal cancer cells [51]. HeLa cells with reduced levels of NR4A proteins displayed a higher tendency for cell death through anoikis [68]. Circadian rhythm is an endogenous 24-hour cycle consisting of an input pathway, master clock, and an output pathway; the underlying mechanism of rhythmistic control is conserved across species [69]. The clock regulates biological processes in a temporal manner by synchronising peripheral oscillators possibly through glucocorticoids, enabling the adaptation and synchronisation of hormones, sleep-wake cycles, and daily activities with changing environmental cues [69,70,71]. Recent findings have linked nutrient and energy metabolism to circadian rhythm [54,72]. Genes involved in metabolism such as NR4A family are known to oscillate in liver and muscle [54]. The circadian rhythm can be modulated by external signals (light, food, temperature), and these signals are conveyed through the MAPK and cAMP-signaling pathways [70,73]. Interestingly, central and peripheral oscillators are sensitive to entrainment by light (photic) and food (non-photic), respectively [73,74,75]. The phase-resetting signals provided through food on peripheral clocks are inhibited by glucocorticoids [76]. Interestingly, expression of NR4A2 and Rasd1 are known to be repressed and upregulated by glucocorticoids, respectively [17,61]. Evidence provided by Rasd1 knockout mice show that Rasd1 may be involved in the input pathway of the circadian rhythm by enhancing photic response and reducing the stimulus provided from non-photic inputs [77]. This suggests that Rasd1 might function as the bridging molecule for glucocorticoids to inhibit the phase-resetting pulses by food on peripheral clocks. Rasd1 may then work with NonO to repress genes involved in metabolism activated via the cAMP pathway, which results in selective repression of a subset of target genes. Hence, modulation of NR4A1 and NR4A2 expression by Rasd1 and NonO could have a major impact on the circadian control, and disruption of this process can give rise to metabolic diseases and cancer development [78,79,80,81]. Materials and Methods Plasmid constructs For information on all primer sequences used for cloning, please refer to Table S1. Coding sequence of mouse Rasd1 (843 bp) was amplified from mouse brain cDNA library PACT2 and cloned in frame via restriction sites KpnI and XhoI into expression vectors – pcDNA4/HisMax B (V864-20, Invitrogen, USA, CA), and pXJGST vector modified from parent plasmid pXJ FLAG [82] by replacing the FLAG coding sequence with the GST coding sequence – and designated as pHis-Rasd1 and pGST-Rasd1, respectively. pHA-Rasd1 was constructed by insertion of a HA-tag at the 39 end of the coding sequence of Rasd1and cloned into pcDNA4/HisMax B via KpnI and XhoI. Mouse clone of NonO (1.4 kb) and its truncated constructs were amplified from MGC-6432 (ATCC, USA, VA), and cloned in frame via restriction sites KpnI and XhoI into pcDNA3.1/V5-His B (V810-20, Invitrogen, USA, CA) and pXJGST vectors. PCR-based, site-directed mutagenesis was used to construct all other mutants of NonO and Rasd1, which were cloned into pcDNA3.1/V5-His B, pXJGST and pcDNA4/HisMax B vectors. For NONO knockdown studies, NONO-shRNA was constructed by cloning of the oligonucleotide that targets mRNA of NONO into pSUPER.puro (VEC-PBS- 0007/0008, OligoEngine, USA, WA) vector. The negative control, Neg-shRNA, was constructed by jumbling up the sequence of the oligonucleotide that was used for cloning of NONO-shRNA. Annealed oligonucleotides were cloned in pSU- PER.puro via BglII and HindIII sites. BLAST was performed to ensure specificity of NONO-shRNA, and that Neg-shRNA did not target any non-specific sequences. The annealing process was performed in the annealing buffer (100mM NaCl and 50mM Hepes, pH 7.4) in BioRad PCR machine: 90uC for 4min, 70uC for 10min, and ramped to 37uC over a period of 45min, kept constant at 37uC for 15min, and ramped to 10uC over a period of 45min. Cell culture and transient transfection HEK293T (ATCC CRL-11268), PC-12 (ATCC CRL-1721), and COS-7 (ATCC CRL-1651) cells were cultured as previously described [26,83]. All transfections were performed the next day PLoS ONE | www.plosone.org 10 September 2011 | Volume 6 | Issue 9 | e24401