P19 H-Ras Induces G1/S Phase Delay Maintaining Cells in a Reversible Quiescence State

This is an open-access article distributed under the terms of the Creative Commons Attribution License.[Background]: Three functional c-ras genes, known as c-H-ras, c-K-ras, and c-N-ras, have been largely studied in mammalian cells with important insights into normal and tumorigenic cellular signal transduction events. Two K-Ras mRNAs are obtained from the same pre-mRNA by alternative splicing. H-Ras pre-mRNA can also be alternatively spliced in the IDX and 4A terminal exons, yielding the p19 and p21 proteins, respectively. However, despite the Ras gene family’s established role in tumorigenic cellular signal transduction events, little is known about p19 function. Previous results showed that p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multi-protein complexes in different signaling pathways (Cancer Res 2003, 63 p5178). This observation suggests that p19 and p21 play differential and complementary roles in the cell.[Principal Findings]: We found that p19 regulates telomerase activity through its interaction with p73a/b proteins. We also found that p19 overexpression induces G1/S phase delay; an observation that correlates with hypophosphorylation of both Akt and p70SK6. Similarly, we also observed that FOXO1 is upregulated when p19 is overexpressed. The three observations of (1) hypophosphorylation of Akt, (2) G1/S phase delay and (3) upregulation of FOXO1 lead us to conclude that p19 induces G1/S phase delay, thereby maintaining cells in a reversible quiescence state and preventing entry into apoptosis. We then assessed the effect of p19 RNAi on HeLa cell growth and found that p19 RNAi increases cell growth, thereby having the opposite effect of arrest of the G1/S phase or producing a cellular quiescence state.[Significance]: Interestingly, p19 induces FOXO1 that in combination with the G1/S phase delay and hypophosphorylation of both Akt and p70SK6 leads to maintenance of a reversible cellular quiescence state, thereby preventing entry into apoptosis.This work was supported by Fundacion de Investigacion Medica Mutua Madrileña Automovilista (Fundacion MMA), the Plan Nacional (MEC) BFU2005-00701 and the Fundacion Eugenio Rodriguez Pascual. M.C. was a recipient of a Fmed MMA fellowship.Peer reviewe

Downregulation of p68 RNA helicase (DDX5) activates a survival pathway involving mTOR and MDM2 signals

The DEAD box p68 RNA helicase (DDX5) is required to manipulate RNA structures implicated in mRNA/rRNA processing and transcript export, and acts as a co-activator for a range of transcription factors. Previous research has indicated that p68 RNA helicase may also be important in tumour development. Wild-type HeLa and stable HeLa (clone 13) cell cultures containing RNAi-mediated depletion of p68 RNA helicase induced by doxycycline (DOX) were used to study how the p68 RNA helicase affects the mTOR cell signalling pathway. Relevant results were repeated using transient transfection with pSuper/pSuper-p68 RNA helicase, containing RNAi-mediated depletion of p68 RNA helicase, to avoid DOX interference. Here we provide strong evidence for the participation of p68 RNA helicase in mTOR regulation. In detail, depletion of this helicase decreases cell growth and activates the mTOR/ MDM2 cell survival mechanism, which ultimately leads to inhibition of the pro-apoptotic activity. p68 RNA helicase downregulation strongly stimulates 4E-BP1 phosphorylation, thereby provoking activation of cap-dependent translation. In contrast, the IRES-dependent translation of c-myc is reduced when p68 RNA helicase is depleted, thus indicating that at least this specific translation requires p68 RNA helicase activity to manipulate the complex 5' end of this mRNA. Interestingly, p68 RNA helicase depletion decreases cell growth while activating the mTOR/MDM2 cell survival mechanism. As MDM2 is a known negative regulator of p53, we infer that the activation of the cell survival mechanism may result in inhibition of the pro-apoptotic factor p53. Finally, p68 RNA helicase depletion activates capdependent translation and inhibits c-MYC IRES-mediated translationThis work was supported by the Fundación Eugenio Rodriguez Pascual, the Plan Nacional (MEC) BFU2005-00701 and FIS PI080007.Peer reviewe

P68 RNA Helicase (DDX5) Alters Activity of Cis- and Trans-Acting Factors of the Alternative Splicing of H-Ras

Background: H-Ras pre-mRNA undergoes an alternative splicing process to render two proteins, namely p21 H-Ras and p19 H-Ras, due to either the exclusion or inclusion of the alternative intron D exon (IDX), respectively. p68 RNA helicase (p68) is known to reduce IDX inclusion. Principal Findings: Here we show that p68 unwinds the stem-loop IDX-rasISS1 structure and prevents binding of hnRNP H to IDX-rasISS1. We also found that p68 alters the dynamic localization of SC35, a splicing factor that promotes IDX inclusion. The knockdown of hnRNP A1, FUS/TLS and hnRNP H resulted in upregulation of the expression of the gene encoding the SC35-binding protein, SFRS2IP. Finally, FUS/TLS was observed to upregulate p19 expression and to stimulate IDX inclusion, and in vivo RNAi-mediated depletion of hnRNP H decreased p19 H-Ras abundance. Significance: Taken together, p68 is shown to be an essential player in the regulation of H-Ras expression as well as in a vital transduction signal pathway tied to cell proliferation and many cancer processes