Involvement of the specific nucleolar protein SURF6 in regulation of proliferation and ribosome biogenesis in mouse NIH/3T3 fibroblasts

The nucleolar proteins which link cell proliferation to ribosome biogenesis are regarded to be potentially oncogenic. Here, in order to examine the involvement of an evolutionary conserved nucleolar protein SURF6/Rrp14 in proliferation and ribosome biogenesis in mammalian cells, we established stably transfected mouse NIH/3T3 fibroblasts capable of conditional overexpression of the protein. Cell proliferation was monitored in real-time, and various cell cycle parameters were quantified based on flow cytometry, Br-dU-labeling and conventional microscopy data. We show that overexpression of SURF6 accelerates cell proliferation and promotes transition through all cell cycle phases. The most prominent SURF6 pro-proliferative effects include a significant reduction of the population doubling time, from 19.8 ± 0.7 to 16.2 ± 0.5 hours (t-test, p < 0.001), and of the length of cell division cycle, from 17.6 ± 0.6 to 14.0 ± 0.4 hours (t-test, p < 0.001). The later was due to the shortening of all cell cycle phases but the length of G1 period was reduced most, from 5.7 ± 0.4 to 3.8 ± 0.3 hours, or by ～30%, (t-test, p < 0.05). By Northern blots and qRT-PCR, we further showed that the acceleration of cell proliferation was concomitant with an accumulation of rRNA species along both ribosomal subunit maturation pathways. It is evident, therefore, that like the yeast homologue Rrp14, mammalian SURF6 is involved in various steps of rRNA processing during ribosome biogenesis. We concluded that SURF6 is a novel positive regulator of proliferation and G1/S transition in mammals, implicating that SURF6 is a potential oncogenic protein, which can be further studied as a putative target in anti-cancer therapy.     

Yeast 3-phosphoinositide-dependent protein kinase-1 (PDK1) orthologs Pkh1-3 differentially regulate phosphorylation of protein kinase A (PKA) and the protein kinase B (PKB)/S6K ortholog Sch9

Pkh1, -2, and -3 are the yeast orthologs of mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1). Although essential for viability, their functioning remains poorly understood. Sch9, the yeast protein kinase B and/or S6K ortholog, has been identified as one of their targets. We now have shown that in vitro interaction of Pkh1 and Sch9 depends on the hydrophobic PDK1-interacting fragment pocket in Pkh1 and requires the complementary hydrophobic motif in Sch9. We demonstrated that Pkh1 phosphorylates Sch9 both in vitro and in vivo on its PDK1 site and that this phosphorylation is essential for a wild type cell size. In vivo phosphorylation on this site disappeared during nitrogen deprivation and rapidly increased again upon nitrogen resupplementation. In addition, we have shown here for the first time that the PDK1 site in protein kinase A is phosphorylated by Pkh1 in vitro, that this phosphorylation is Pkh-dependent in vivo and occurs during or shortly after synthesis of the protein kinase A catalytic subunits. Mutagenesis of the PDK1 site in Tpk1 abolished binding of the regulatory subunit and cAMP dependence. As opposed to PDK1 site phosphorylation of Sch9, phosphorylation of the PDK1 site in Tpk1 was not regulated by nitrogen availability. These results bring new insight into the control and prevalence of PDK1 site phosphorylation in yeast by Pkh protein kinases.     

The plant alkaloid chelerythrine binds to chromatin,alters H3K9Ac and modulates global gene expression

Chelerythrine (CHL), a plant alkaloid, possesses antimicrobial, anti-inflammatory, and antitumor properties. Although CHL influences several key signal transduction pathways, its ability to interact directly with nucleoprotein complex chromatin, in eukaryotic cells has so far not been looked into. Here we have demonstrated its association with hierarchically assembled chromatin components, viz. long chromatin, chromatosome, nucleosome, chromosomal DNA, and histone H3 and the consequent effect on chromatin structure. CHL was found to repress acetylation at H3K9. It is more target-specific in terms of gene expression alteration and less cytotoxic compared to its structural analog sanguinarine.     

CTR9/PAF1c regulates molecular lineage identity,histone H3K36 trimethylation and genomic imprinting during preimplantation development

Genome-wide epigenetic reprogramming is required for successful preimplantation development. Inappropriate or deficient chromatin regulation can result in defective lineage specification and loss of genomic imprinting, compromising normal development. Here we report that two members of the RNA polymerase II associated factor, homolog (Saccharomyces cerevisiae) complex (PAF1 complex) components, Ctr9 and Rtf1, are required during mammalian preimplantation development. We demonstrate that Ctr9-deficient embryos fail to correctly specify lineages at the blastocyst stage. Expression of some lineage specific factors is markedly reduced in Ctr9 knockdown embryos, including Eomes, Elf5 and Sox2, while others are inappropriately expressed (Oct4, Nanog, Gata6, Fgf4 and Sox17). We also show that several imprinted genes (Mest, Peg3, Snrpn and Meg3) are aberrantly expressed although allele specific DNA methylation is not altered. We document a loss of histone H3 lysine 36 trimethylation (H3K36me3) in Ctr9-deficient embryos and confirm that knockdown of either Setd2 or Rtf1 results in similar phenotypes. These findings show that the PAF1 complex is required for mammalian development, likely through regulation of H3K36me3, and indicate functional conservation of the PAF1 complex from yeast to mammals in vivo.     

Identification of gibberellin A4 in Pisum sativum L. and the effects of applied gibberellins A9, A4, A5 and A3 on the le mutant

Gibberellin A₄ (GA₄) was identified for the first time in the garden pea (Pisum sativum) L.), by gas chromatography-mass spectrometry. However, in wild-type shoots the level of GA₄ was only about 6% of the level of GA₁, and it is therefore unlikely that GA₄ plays a major role per se in the control of pea stem elongation. In shoots of the le mutant, GA₄ was not detected, while the level of GA₉ was approximately twice that found in the wild-type. The le mutation also markedly reduced the elongation response to applied GA₉. It appears, therefore, that in Pisum the le mutation blocks the 3-hydroxylation of GA₉ to GA₄, in addition to the 3-hydroxylation of GA₂₀ to GA₁. In contrast, the le mutation did not reduce the response to applied GA₅, suggesting the step GA₅ to GA₃ is not catalysed by the enzyme controlled by the Le gene. The step GA₅ to GA₃ was confirmed in peas by metabolite analysis after treatment with deuterated GA₅.     

Synthesis and characterization of cis-[(PPh3)2Pt{9-MeAd(-H),NN}]X (X = NO3, PF6): The first example of a platinum(II) complex containing the N,N-chelated 9-methyladenine anion

Reaction between the binuclear hydroxo complex cis-[(PPh₃)₂Pt(μ-OH)]₂X₂ (X⁻ = NO₃, 1a; , 1b) and the model DNA base 9-methyladenine (9-MeAd) leads to the formation of the mononuclear species cis-[(PPh₃)₂Pt{9-MeAd(-H),N⁶N⁷}]X (X⁻ = NO₃, 2a; PF₆, 2b), in which the nucleobase chelates the Pt(II) ion with the N6 and N7 atoms. The coordination mode of the nucleobase has been determinated through a multinuclear (¹H, ³¹P, ¹³C, ¹⁵N and ¹⁹⁵Pt) NMR analysis and the nuclearity of the complex has been obtained by E.S.I. mass spectrometry. 2 represents the first example of an isolated platinum complex in which the NH₂-deprotonated adenine exhibits this binding mode.