Rasd1 Modulates the Coactivator Function of NonO in the Cyclic AMP Pathway

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

Molecular study of Rasd1 transcriptional regulation and Rasd1-NonO interaction.

RAS, Dexamethasone-induced 1 (Rasd1) is a monomeric guanosine 5’-triphosphate (GTP) binding protein of the Ras subfamily involved in diverse physiological roles including circadian rhythm, stress and iron homeostasis. In this study, transcriptional regulation of Rasd1 and identification of novel interacting partner of Rasd1 are investigated to better understand how Rasd1 performs these physiological functions. The Rasd1 minimal promoter is identified to reside within 111 bp upstream of Rasd1 via study of the Rasd1/luciferase fusion constructs. Subsequent studies using site-directed mutagenesis, reporter gene fusion assays, gene knockdown, chromatin immunoprecipitation (ChIP) and Real-Time (RT-PCR) experiments show that transactivators, D site of albumin promoter binding protein (Dbp; transcription factor involved in the output pathway of circadian rhythm) and glucocorticoid receptor (GR; transactivates Rasd1 upon Dexamethasone (Dex; synthetic glucocorticoid) induce Rasd1 promoter activity. I also find that Dbp enhances Rasd1 promoter activity by binding to the CCAAT/enhancer binding protein beta (C/EBPβ) site located at the 5’ untranslated region (UTR) of Rasd1. Circadian expression of Rasd1 requires promoter sequence of Rasd1 and downstream GRE elements and is modulated by Dbp. In addition, my studies identify NonO, Non-POU domain-containing octamer-binding protein, as a novel Rasd1 interacting partner, which is involved in regulation of cyclic adenosine monophosphate (cAMP) pathway, circadian rhythm, RNA processing and transcription. Interaction of Rasd1 and NonO is validated with affinity pulldowns, co-IP assays and indirect immunofluorescence. Investigation via reporter gene assays, ChIP and gene knockdown suggest that Rasd1 modulates NonO to function as a co-repressor of the cAMP pathway by interacting at the cAMP response element (CRE) site of specific target genes – nuclear orphan receptor 4A (NR4A) 1 and 2. The GTP hydrolysis activity of Rasd1 is required for the functional interaction. These findings reveal a novel mechanism by which the coregulator activity of NonO can be modulated.​Doctor of Philosophy (SBS

Rasd1 interacts with Ear2 (Nr2f6) to regulate renin transcription

The Rasd1 protein is a dexamethasone induced monomeric Ras-like G protein that oscillates in the suprachiasmatic nucleus (SCN). Previous studies have shown that Rasd1 modulates multiple signaling cascades. However, it is still unclear exactly how Rasd1 carries out its function. Studying protein-protein interactions involving Rasd1 may provide insights into its biological functions in different contexts. Results: To further explore the molecular function of Rasd1, we performed a yeast two-hybrid screen and identified Ear2, a negative regulator of renin transcription, as an interaction partner of Rasd1. We validated the interaction in vitro and in transfected COS-7 cells. We further confirmed the interaction of endogenous Rasd1 and Ear2 from HEK293T cell and mouse brain extract. Rasd1 inhibited transcriptional repression by Ear2 on a renin promoter-luciferase reporter construct both in the presence and absence of all-trans-retinoic acid. Moreover, realtime RT-PCR showed upregulation of endogenous renin transcription in As4.1 cells over-expressing Rasd1. We demonstrated that the ligand binding domain of Ear2 is required for physical and functional interaction between the two proteins. In addition, we demonstrated that shRNA-mediated knockdown of Rasd1 results in further repression of Ear2-mediated renin transcription, whereas induction of Rasd1 by dexamethasone counteracts the effects of shRNA-mediated Rasd1 knockdown. Finally, our study showed that Rasd1 missense mutations not only attenuate their physical interaction with Ear2 but also abolish their ability to counteract repression of renin transcription mediated by Ear2. Conclusions: Our study provides evidence for physical and functional interactions between Rasd1 and Ear2. The results suggest that their interactions are involved in renin transcriptional regulation. These findings not only reveal a novel role for Rasd1-medated signaling but also provide the basis for potential intervention of renin expression.Published versio