Rasd1 Modulates the Coactivator Function of NonO Rasd1 in the event of single transfection (Figures A 5, 12, 19, 26, and 33). However, co-transfection of plasmids expressing Rasd1 mutants and NonO did not affect the mutants’ sub-cellular distribution, unlike that of wild-type Rasd1 (compare Figures A5 with A 15, 22, 29, and 36). Likewise, the sub- cellular distribution of NonO was also unaffected by the presence of Rasd1 mutants, A178V, G81A and Del-NLS (compare Figures A2 with A 16, 23, and 37). Interestingly, NonO was translocated to the cytoplasm in the presence of T38N (compare Figures A2 with A30). (B) Effects of Rasd1 mutants on the CREB pathway in the presence of NonO were studied in HEK293T cells. Cells were co-transfected with pNonO-V5 and either Rasd1 or Rasd1 mutants, and luciferase assays were performed after lysis of cells. Cells were induced with forskolin for 4 hours prior to harvesting. The CREB- mediated transcription was repressed in cells co-transfected with pNonO-V5 (2 mg) and pHis-Rasd1 (2 mg) (Compare Bars II with III). However, the repressive effect on CREB is abolished in cells transfected with Rasd1 mutants expressing plasmids compared with cells transfected with pHis-Rasd1 (Compare Bars III with IV–VII). ‘*’ – p,0.05; ‘**’ – p,0.01; ‘***’ – p,0.001. (C) Interaction studies of NonO and Rasd1 mutants were studied via co- transfection of pNonO-V5 and pGST-Rasd1 mutant clones in COS-7 cells. GST-pulldown was subsequently performed and similar to wild-type, only constructs T38N and A178V were able to interact with NonO (Lanes 2, 3 and 5). doi:10.1371/journal.pone.0024401.g003 energy dependent and requires GTP-bound Rasd1. Further studies will be required to decipher the exact mechanism employed by NonO to enable the enhanced nuclear presence of Rasd1. Recently, Chuderland et al. identified a novel nuclear localisation sequence termed the NTS (nuclear translocation sequence), composed of phosphorylated S/T-P-S/T, that enables nuclear translocation of the protein via binding to importin 7 [38,39]. This mode of nuclear translocation is used by proteins of different signaling pathways, including the ERK (extracellular signal-regulated kinase) pathway, and was initially discovered on ERK-2 [38,39]. This mode of nuclear entry enables rapid response to signaling and also adequately explains the increase in the amount of Rasd1 found in the nucleus in the presence of NonO. Interestingly, a ‘TPT’ amino acid sequence is found in Rasd1; this sequence is evolutionarily conserved, and is also present in Rasd2, a paralog of Rasd1. Further studies are required to determine if nuclear translocation of Rasd1 requires its ‘TPT’ sequence. In our study, we observed that NonO only upregulates CREB- GAL4DBD fusion protein activity slightly via reporter gene assay. This observation contradicts a previous study, which suggests that NonO works as a strong co-activator of the cAMP-dependent pathway via interaction with TORC2, tranducers of the regulated CREB [13]. In our reporter gene system, the CREB-GAL4DBD fusion construct used in the assay lacks the bZIP domain. It has been shown that TORC2 interacts with CREB via the bZIP domain [40]. Hence, deletion of the bZIP domain in the CREB- GAL4DBD construct prevents interaction between CREB and TORC2 [8]. This may serve to explain the minor induction of the luciferase activity upon transfection of NonO in HEK293T cells. Many transcription factors, RNA-binding proteins and tran- scriptional co-regulators are known to be bi- or multi-functional proteins. Some bi-functional proteins, including CoAA, PGC-1, CAPERa and CAPERb, and steroid receptors, are shown to be involved in transcription co-activation and alternative splicing [15,41,42,43]. Multi-functional proteins like NONO and PSF (polypyrimidine tract-binding protein-associated splicing factor) perform RNA processing functions, transcriptional activation and repression, and RNA transport [12,13,14,15,31,32,33,44,45]. In addition, NONO is also involved in circadian rhythm as an antagonist of Per1 [46]. Multiple lines of evidence indicate that NonO is a multi-tasking protein with bimodal function in transcription [12,13,14,15,31,32,33,46]. NonO is known to serve as a co-activator by interacting with TORC2 to up-regulate target genes of the cAMP-dependent pathway [13]. However, NonO is also known to repress transcription by recruiting histone deacetylase (HDAC) to the target promoter by itself or via interaction with PSF [12,15]. In addition, NonO is known to interact directly with histone to suppress transcription of prolyl-4- hydroxylase a1 upon induction of cells with TNFa [15]. Moreover, NonO has been shown to serve as both co-activator and repressor of androgen receptor-regulated gene transcription depending on the other proteins associated with the transcriptional initiation complex [44,45]. Interestingly, another interacting partner of NonO, DJ-1, has been shown to switch NonO from a co-repressor to a co-activator for neuroprotection functions [47]. In this study, we show that binding of Rasd1 to NonO signals NonO to switch from a co-activator to co-repressor mode to suppress transcription of a subset of the CREB target genes. Our case is similar to NonO’s regulation of the androgen receptor where NonO can either activate or repress transcription of the androgen receptor depending on the proteins associated with it [44,45]. Since Rasd1 is known to serve as a transcriptional co- repressor of FE-65 [19], and as an antagonist to the function of transcription factor, Ear-2, in the repression of Renin’s transcrip- tion [34]; it is conceivable that Rasd1 might enable NonO to serve as a transcriptional co-repressor of the CREB signaling pathway. We observed that co-transfection of pNonO-V5 and pGST-Rasd1 resulted in a substantial increase in the nuclear localisation of Rasd1. This finding resembles that of Lau et al [19], where co- transfection of Rasd1 and FE-65 results in an increased nuclear distribution of Rasd1, and suggests that nuclear translocation of Rasd1 is required for suppression of target-gene transcription. In addition, we observed that Rasd1 and NonO co-suppress the transcription of a subset of CREB target genes. It is intriguing that, unlike NR4A1 and NR4A2 transcripts, FOS transcription was unaffected by Rasd1 and NonO. The discrepancy observed might be attributed to the differences in the mechanisms involved in the transcriptional regulation of NR4A and FOS. Transcriptional regulation of FOS takes place at both transcriptional initiation and elongation processes, which allows an additional level of control of the FOS gene [48,49,50]. Currently, NR4A proteins are only known to be regulated by transcription factors at the transcription initiation stage [13,51,52]. Our study also suggests that Rasd1 and NonO bind the CRE-site of the NR4A2 promoter to repress its transcription and that the mechanism employed by Rasd1 and NonO in the repression of transcription of NR4A genes involves the regulation of proteins required for the transcription initiation step. Further studies will need to be performed to explore the mechanism involved. Many signal transduction pathways converge in the nucleus through modulating CREB, whose phosphorylation pattern influences binding of its co-activators, including CBP/p300 in the presence or absence of TORC2 [7,8]. Phosphorylation of CREB by PKA and other kinases of the cAMP signaling pathway activates CREB to recruit co-activators, other transcription factors, and general transcription factors to the target promoter, resulting in the transactivation of target genes [13]. It remains unclear how CREB is able to converge diverse signals and elicit differential effects on target gene expression. In the case of the cAMP-dependent pathway, the co-regulators that interact with CREB may play an important role for the cell to have a specific response in different contexts [30]. There are a plethora of CREB co-regulators, and their activities are known to be regulated by PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24401