The role of p19 and p21 H-Ras proteins and mutants in miRNA expression in cancer and a Costello syndrome cell model.

BACKGROUND: P19 H-Ras, a second product derived from the H-Ras gene by alternative splicing, induces a G1/S phase delay, thereby maintaining cells in a reversible quiescence state. When P21 H-Ras is mutated in tumour cells, the alternative protein P19 H-Ras is also mutated. The H-Ras mutation Q61L is frequently detected in different tumours, which acts as constitutive activator of Ras functions and is considered to be a strong activating mutant. Additionally, a rare congenital disorder named Costello Syndrome, is described as a H-Ras disorder in children, mainly due to mutation G12S in p19 and p21 H-Ras proteins, which is present in 90 % of the Costello Syndrome patients. Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression. METHODS: Total miRNAs expression regulated by H-Ras proteins were first analyzed in human miRNA microarrays assays. Previously selected miRNAs, were further analyzed in developed cell lines containing H-Ras protein mutants, that included the G12S Costello Syndrome mutant, with PCR Real-Time Taq Man miRNA Assays primers. RESULTS: This study describes how p19 affects the RNA world and shows that: i) miR-342, miR-206, miR-330, miR-138 and miR-99b are upregulated by p19 but not by p19W164A mutant; ii) anti-miR-206 can restore the G2 phase in the presence of p19; iii) p19 and p21Q61L regulate their own alternative splicing; iv) miR-206 and miR-138 are differentially regulated by p19 and p21 H-Ras and v) P19G12S Costello mutants show a clear upregulation of miR-374, miR-126, miR-342, miR-330, miR-335 and let-7. CONCLUSIONS: These results allow us to conclude that the H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome. Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers.

P68 RNA Helicase (DDX5) Required for the Formation of Various Specific and Mature miRNA Active RISC Complexes.

INTRODUCTION: DEAD-box RNA helicases catalyze the ATP-dependent unwinding of doublestranded RNA. In addition, they are required for protein displacement and remodelling of RNA or RNA/protein complexes. P68 RNA helicase regulates the alternative splicing of the important protooncogene H-Ras, and numerous studies have shown that p68 RNA helicase is probably involved in miRNA biogenesis, mainly through Drosha and RISC/DICER complexes. OBJECTIVE: This study aimed to determine how p68 RNA helicase affects the activity of selected mature miRNAs, including miR-342, miR-330, miR-138 and miR-206, miR-126, and miR-335, and let-7a, which are known to be related to cancer processes. METHODS: The miRNA levels were analyzed in stable HeLa cells containing p68 RNA helicase RNAi induced by doxycycline (DOX). Relevant results were repeated using transient transfection with pSuper/ pSuper-p68 RNA helicase RNAi to avoid DOX interference. RESULTS: Herein, we reported that p68 RNA helicase downregulation increases the accumulation of the mature miRNAs, such as miR-126, let-7a, miR-206, and miR-138. Interestingly, the accumulation of these mature miRNAs does not downregulate their known protein targets, thus suggesting that p68 RNA helicase is required for mature miRNA-active RISC complex activity. CONCLUSION: Furthermore, we demonstrated that this requirement is conserved, as drosophila p68 RNA helicase can complete the p68 RNA helicase depleted activity in human cells. Dicer and Drosha proteins are not affected by the downregulation of p68 RNA helicase despite the fact that Dicer is also localized in the nucleus when p68 RNA helicase activity is reduced.

Roles of hnRNP A1, SR proteins, and p68 helicase in c-H-ras alternative splicing regulation.

Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.

Alternative splicing of the human proto-oncogene c-H-ras renders a new Ras family protein that trafficks to cytoplasm and nucleus.

We characterized a novel protein of the Ras family, p19 (H-RasIDX). The c-H-ras proto-oncogene undergoes alternative splicing of the exon termed IDX. We show that the alternative p19 mRNA is stable and as abundant as p21 (p21 H-Ras4A) mRNA in all of the human tissues and cell lines tested. IDX is spliced into stable mRNA in different mammalian species, which present a high degree of nucleotide conservation. Both the endogenous and the transiently expressed p19 protein are detected in COS-1 and HeLa cells and show nuclear diffuse and speckled patterns as well as cytoplasmic localization. In yeast two-hybrid assays, p19 did not interact with two known p21 effectors, Raf1 and Rin1, but was shown to interact with RACK1, a scaffolding protein that promotes multiprotein complexes in different signaling pathways. This observation suggests that p19 and p21 play differential and complementary roles in the cell.

Study of the 2719 mutant of the c-H-ras oncogene in a bi-intronic alternative splicing system.

C-H-ras proto-oncogene forms part of the signal transduction pathway of numerous external stimuli. This proto-oncogene is regulated by alternative splicing within its intron D due to the presence of the alternative intron D exon (IDX). The alternative splicing produces mRNA which encodes for the putative p19 protein, that lacks transforming potential. Herein, we demonstrated that SR proteins regulate the intron D splicing. Moreover, we studied the 2719 mutation of H-ras which has higher transforming potential than Ile12 and Val12 H-ras mutants and is also known to affect the 5' splice site of the IDX. However, here we show that the 2719 mutant can still be spliced when the upstream 5' splice-site is blocked. During these later studies, additionally, we generated a short 11 nucleotides 5' terminal exon that was fully defined and spliced in a bi-intronic pre-mRNA. The definition of this mini-exon was also addressed in this work.

P19 H-ras induces G1/S phase delay maintaining cells in a reversible quiescence state.

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 p73alpha/beta 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.

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.