Rasd1 Modulates the Coactivator Function of NonO in the Cyclic AMP Pathway Shufen Angeline Ong1, Jen Jen Tan1,2, Wai Loon Tew 1 , Ken-Shiung Chen1* 1 School of Biological Sciences, Department of Genomics and Genetics, Nanyang Technological University, Singapore, Singapore, 2 Agency for Science, Technology and Research, Singapore, Singapore Abstract All living organisms exhibit autonomous daily physiological and behavioural rhythms to help them synchronize with the environment. Entrainment of circadian rhythm is achieved via activation of cyclic AMP (cAMP) and mitogen-activated protein kinase signaling pathways. NonO (p54nrb) is a multifunctional protein involved in transcriptional activation of the cAMP pathway and is involved in circadian rhythm control. Rasd1 is a monomeric G protein implicated to play a pivotal role in potentiating both photic and nonphotic responses of the circadian rhythm. In this study, we have identified and validated NonO as an interacting partner of Rasd1 via affinity pulldown, co-immunoprecipitation and indirect immunofluorescence studies. The GTP-hydrolysis activity of Rasd1 is required for the functional interaction. Functional interaction of Rasd1-NonO in the cAMP pathway was investigated via reporter gene assays, chromatin immunoprecipitation and gene knockdown. We showed that Rasd1 and NonO interact at the CRE-site of specific target genes. These findings reveal a novel mechanism by which the coregulator activity of NonO can be modulated. Citation: Ong SA, Tan JJ, Tew WL, Chen K-S (2011) Rasd1 Modulates the Coactivator Function of NonO in the Cyclic AMP Pathway. PLoS ONE 6(9): e24401. doi:10.1371/journal.pone.0024401 Editor: Janet F. Partridge, St. Jude Children’s Research Hospital, United States of America Received June 14, 2011; Accepted August 8, 2011; Published September 7, 2011 Copyright: 2011 Ong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from the Singapore Ministry of Education, Academic Research Fund Tier 2 (ARC 3/04, M45080000) awarded to Ken- Shiung Chen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kschen@ntu.edu.sg Introduction The cAMP-dependent pathway is known to respond to information obtained from numerous extracellular stimuli to regulate processes including synaptic plasticity, neuronal differen- tiation, circadian rhythm, memory, and glucose homeostasis [1,2,3,4,5,6]. Despite the involvement of unique neurotransmitters, hormones or other signals, and different intracellular signaling systems, these pathways all converge at the nucleus. Hence, specificity of the signal and the pathway induced is crucial to ensure that specific proteins are transcribed to perform precise functions in a tissue- and/or temporal-specific manner. This specificity is achieved by the type of signals, how the signals are detected and relayed to specific signaling proteins responding to the stimuli, and the subsequent interactions with other proteins, and is dependent on cell type and contexts. Regulation of the pathway can occur at any step of the signal transduction process but one of the more prominent regulations is at the transcriptional level. Regulation of the pathway at the transcriptional level is achieved by various mechanisms including inhibition of core transcription factor activity, sequestration, and competition for limiting factor [7,8,9]. NonO is predominantly localized in the paraspeckles [10], a sub-compartment of the nucleus, and is a member of the family of RNA-Recognition Motif (RRM) containing proteins [11]. NonO is a co-activator of CREB and has been known to serve in both transcriptional activation and repression [12,13,14,15]. In our current study, NonO is identified as a binding partner of Rasd1, a monomeric G protein belonging to the RAS family [16,17]. Traditionally, RAS proteins function as cytoplasmic signal transducers of diverse intracellular signaling pathways including the cAMP- dependent pathway [16]. Similar to its other family members, Rasd1 harbours a CAAX motif at its C-terminal and displays a high degree of conservation in its G boxes, which are responsible for the guanine nucleotide binding and hydrolysis activities of RAS proteins. Mutations in the G boxes have been shown to disrupt the functions of RAS proteins [16,18,19,20,21,22,23,24]. Rasd1 has been shown in various studies to be involved as signal transducers of multiple signaling pathways, including iron homeostasis, growth hormone secretion and circadian rhythm [20,25,26,27,28,29]. Recently, Rasd1 has also been observed to reside in the nucleus, serving as a transcriptional repressor of glycogen synthase kinase 3b [19] as well as an inhibitor of the cAMP-dependent pathway [20,28,29]. In this study, we identify NonO as a novel binding partner of Rasd1. This is the first study that shows the novel interaction of a RRM-possessing protein with a monomeric G protein. In the nucleus, Rasd1 binds to NonO and regulates the cAMP-dependent pathway at the transcriptional level. GTP-hydrolysis activity of Rasd1 is required for repressing CREB activity. We propose a new mechanism of regulating the cAMP-dependent pathway at the transcriptional level via modulation of the co-activator’s function. Binding of Rasd1 to NonO modulates NonO’s functions by changing NonO from a co-activator to a co-repressor of the cAMP- dependent pathway. Rasd1 and NonO cooperate to suppress the transcription of a subset of CRE-containing genes, NR4A 1 & 2. This finding adds weight to how specificity of signaling pathways is achieved via the usage of different interacting partners to modulate the function of a multi-tasking co-regulator [30]. PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24401