Trimethylguanosine nucleoside inhibits cross-linking between snurportin 1 and m3G-capped U1 snRNA

Macromolecular nuclear import is an energy-and signal-dependent process. The best characterized type of nuclear import consists of proteins carrying the classical NLS that is mediated by the heterodimeric receptor importin α/β. Spliceosomal snRNPs U1, U2, U4, and U5 nuclear import depend both on the 5' terminal m3G (trimethylguanosine) cap structure of the U snRNA and the Sm core domain. Snurportin 1 recognizes the m3G-cap structure of m3G-capped U snRNPs. In this report, we show how a synthesized trimethylguanosine nucleoside affects the binding of Snurportin 1 to m3G-capped U1 snRNA in a UV-cross-linking assay. The data indicated that TMG nucleoside is an essential component required in the recognition by Snurportin 1, thus suggesting that interaction of Snurportin 1 with U1 snRNA is not strictly dependent on the presence of the whole cap structure, but rather on the presence of the TMG nucleoside structure. These results indicate that the free nucleoside TMG could be a candidate to be an inhibitor of the interaction between Snurportin 1 and U snRNAs. We also show the behavior of free TMG nucleoside in in vitro U snRNPs nuclear import. Copyright © Taylor & Francis Group, LLC.This work was supported by Plan Nacional BFU2005-00701 and the Polish Committee for Scientific Research (KBN) # 6 P04A 055 17. D.B. was a recipient of a CNPq Brazilian fellowship and EMBO and FEBS short-term fellowshipsPeer Reviewe

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

Role of the miRNA regulated by the protein p19 H-Ras. Study of the presence of mirtrones within the H-Ras gene

Peer reviewe

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.This work was supported by The Asociación Española contra el Cáncer, La Marató de TV3 and Fundación Ramón Areces and the Polish Committee for Scientific Research (KBN) # 6 P04A 055 17. S Guil was a recipient of a BEFI fellowship.Peer Reviewe

SS-B (La) nuclear antigen: Fast and non-degradative procedure to prepare SS-B extracts free from other nuclear antigens

A procedure is described allowing the easy and fast obtention of a cellular extract from calf thymus, enriched in the undegraded 52 kDa SS-B protein. As seen by western blot, the extract does not contain Sm and RNP antigens, allowing the use of such fraction for the detection of anti-SS-B antibodies without the interference of the anti-Sm and anti-RNP specificities. The enrichment avoids denaturing agents, making the fraction suitable for use in functional and structural studies.This work was supported by grants from CAICYT, Fundacion M. Francisco de Roviralta and Fondo de Investigations Sanitarias.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

G12S oncogenic mutation in c-H-Ras proteins is modulated by p19 protein via NM23H1 in transfected HeLa cell model

Trabajo presentado en la XV Reunión Internacional de Ciencias Médicas, celebrada en León, Guanajuato (México), del 22 al 24 de abril de 2015Peer Reviewe

A monoclonal antibody that specifically recognizes m6A nucleoside

A hybridoma against the nucleoside m6A has been obtained from mouse spleen. This hybridoma was named H65 and it secretes monoclonal antibodies anti-m6A. The competition assays showed that the monoclonal antibody was highly specific for m6A nucleoside.This work was supported by PGC grant no PB92-0004. C. Codony was recipient of a fellowship from PGC.Peer Reviewe

Characterization of antigenic polypeptides of the RNP, Sm and SS-B nuclear antigens from calf thymus

Antinuclear autoantibodies are a hallmark of autoimmune diseases. The RNP, Sm and SS-B nuclear antigens from calf thymus in whole tissue, nuclear extracts and fractions have been studied by using different techniques including immunodiffusion, counterimmunoelectrophoresis and protein blotting. Such studies were done in order to obtain a precise characterization of the polypeptide components of those antigens. From our results it can be well established that: (a) one 69.8 Kd polypeptide (for whole tissue and nuclei) and a number of well-defined 32-38-Kd polypeptides (for nuclear extracts and ammonium sulfate fractions) show an antigenic character against anti-RNP sera; (b) anti-Sm sera from different patients show in all cases a variable component of antigenic polypeptides, including one 28.8, 29.7 Kd doublet and two singlets of 14.8 and 11.0 Kd; and (c) a 52.0-Kd SS-B antigenic polypeptide is found for whole tissue, which is gradually degraded in nuclei and nuclear extracts to a more stable 47.1-Kd polypeptide.N.D. and M.B. are recipients of fellowship from the Fondo de Investigaciones Sanitarias and C.S.I.C. respectively. This work was supported by grants from CAICYT, Fundacion M. Franc&a de Roviralta and Fondo de Investigaciones Sanitarias.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