Expression Preference of the S-adenosylmethionine Synthetase (Sam-S) Gene in Drosophila melanogaster

Ｓ－腺苷甲硫氨酸合成酶（ＳａｍＳ）是目前已知的唯一在生物体内催化腺苷甲硫氨酸合成的酶。它是除自身以外、所有甲基化反应的甲基供体,并且参与多胺的生物合成。多胺对于稳定ＤＮＡ、ＲＮＡ和蛋白质大分子的双螺旋结构具有重要作用,和ＤＮＡ的甲基化一起参与了基因组的印迹（ｉｍｐｒｉｔｉｎｇ）过程。在分离到ＳａｍＳ基因的基础上,本文通过Ｎｏｒｔｈｅｒｎｂｌｏｔ和酶活两种方法,对该基因在野生型果蝇和四个等位突变体发育过程中主要阶段的转录和转译水平进行了测定。野生型果蝇由瑞典Ｕｍｅａ大学果蝇中心提供。由于该基因的突变是阴性致死突变,研究中采用了杂合子突变体：Ｓｕ（ｚ）５,Ｌ（２）Ｍ６,Ｌ（２）Ｒ２３和Ｄｆ（２Ｌ）ＰＭ４４,均由所在实验室诱变获得。Ｎｏｒｔｈｅｒｎ分析时,以ｃＤＮＡ＃１０和ａ微管蛋白基因为探针,分析果蝇卵巢、幼虫、蛹、胚胎、雄性和雌性成蝇中该基因Ｐｏｌｙ（Ａ）ＲＮＡ的转录水平。通过测定蛋白粗提物中的酶活,分析果蝇卵巢、幼虫、蛹、以及雄性成蝇腹部组织中该基因的翻译水平。Ｆｉｇ．１,２＆３表明：ＳａｍＳ基因主要在成熟雌蝇的卵巢中高表达,在雄性成蝇中该基因的表达水平明显低于雌性。在其它发育阶段及组织部位中仅维持     

The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of motor neurons in patients with null mutations in the SMN1 gene. An almost identical SMN2 gene is unable to compensate for this deficiency because a single C‐to‐T transition at position +6 in exon‐7 causes skipping of the exon by a mechanism not yet fully elucidated. We observed that the C‐to‐T transition in SMN2 creates a putative binding site for the RNA‐binding protein Sam68. RNA pull‐down assays and UV‐crosslink experiments showed that Sam68 binds to this sequence. In vivo splicing assays showed that Sam68 triggers SMN2 exon‐7 skipping. Moreover, mutations in the Sam68‐binding site of SMN2 or in the RNA‐binding domain of Sam68 completely abrogated its effect on exon‐7 skipping. Retroviral infection of dominant‐negative mutants of Sam68 that interfere with its RNA‐binding activity, or with its binding to the splicing repressor hnRNP A1, enhanced exon‐7 inclusion in endogenous SMN2 and rescued SMN protein expression in fibroblasts of SMA patients. Our results thus indicate that Sam68 is a novel crucial regulator of SMN2 splicing.     

Exploring the multifaceted roles of heat shock protein B8 (HSPB8) in diseases

HSPB8 is a member of ubiquitous small heat shock protein (sHSP) family, whose expression is induced in response to a wide variety of unfavorable physiological and environmental conditions. Investigation of HSPB8 structure indicated that HSPB8 belongs to the group of so-called intrinsically disordered proteins and possesses a highly flexible structure. Unlike most other sHSPs, HSPB8 tends to form small-molecular-mass oligomers and exhibits substrate-dependent chaperone activity. In cooperation with BAG3, the chaperone activity of HSPB8 was reported to be involved in the delivery of misfolded proteins to the autophagy machinery. Through this way, HSPB8 interferes with pathological processes leading to neurodegenerative diseases. Accordingly, published studies have identified genetic links between mutations of HSPB8 and some kind of neuromuscular diseases, further supporting its important role in neurodegenerative disorders. In addition to their anti-aggregation properties, HSPB8 is indicated to interact with a wide range of client proteins, modulating their maturations and activities, and therefore, regulates a large repertoire of cellular functions, including apoptosis, proliferation, inflammation and etc. As a result, HSPB8 has key roles in cancer biology, autoimmune diseases, cardiac diseases and cerebral vascular diseases.     

Photosynthesis research: advances through molecular biology – the beginnings, 1975–1980s and on...

Restriction endonuclease recognition sites and genes for rRNAs were first mapped on chloroplast chromosomes in 1975–1976. This marked the beginning of the application of molecular biology tools to photosynthesis research. In the first phase, knowledge about proteins involved in photosynthesis was used to identify plastid and nuclear genes encoding these proteins on cloned segments of DNA. Soon afterwards the DNA sequences of the cloned genes revealed the full primary sequences of the proteins. Knowledge of the primary amino acid sequences provided deeper understanding of the functioning of the protein and interactions among proteins of the photosynthetic apparatus. Later, as chloroplast DNA sequencing proceeded, genes were discovered that encoded proteins that had not been known to be part of the photosynthetic apparatus. This more complete knowledge of the composition of reaction centers and of the primary amino acid sequences of individual proteins comprising the reaction centers opened the way to determining the three-dimensional structures of reaction centers. At present, the availability of cloned genes, knowledge of the gene sequences and systems developed to genetically manipulate photosynthetic organisms is permitting experimental inquiries to be made into crucial details of the photosynthetic process. This revised version was published online in August 2006 with corrections to the Cover Date.     

Airway constrictor effects of leukotriene D4 in dogs with hyperreactive airways

Leukotriene D₄ (5 μg/ml) aerosol constricts airways of dogs with nonspecific airway hyperreactivity but not of mongrel dogs which lack nonspecific airway hyperreactivity. R_L increased 200 + 25% and C_dyn decreased to 77 ± 5% of the pre-challenge value. LTD₄ (10 μg/ml) produced no further increase. Atropine (0.2 mg/kg) prevented the increase in R_L and decrease in C_dyn, suggesting that part of the effect of LTD₄ on airways is neurally mediated.     

Sam68 relocalization into stress granules in response to oxidative stress through complexing with TIA-1

Sam68 has been implicated in a variety of important cellular processes such as RNA metabolism and intracellular signaling. We have recently shown that Sam68 cytoplasmic mutants induce stress granules (SG) and inhibit HIV-1 nef mRNA translation [J. Henao-Mejia, Y. Liu, I.W. Park, J. Zhang, J. Sanford, J.J. He, Suppression of HIV-1 Nef translation by Sam68 mutant-induced stress granules and nef mRNA sequestration, Mol. Cell 33 (2009) 87-96]. These findings prompted us to investigate the possibility and the underlying mechanisms of the wild-type counterpart Sam68 SG recruitment. Herein, we revealed that Sam68 was significantly recruited into cytoplasmic SG under oxidative stress. We then demonstrated that domain aa269-321 and KH domain were both essential for this recruitment. Nevertheless, Sam68 knockdown had no effects on SG assembly, indicating that Sam68 is not a constitutive component of the SG. Moreover, we showed that Sam68 cytoplasmic mutant-induced SG formation was independent of eIF2α phosphorylation. Lastly, we demonstrated that Sam68 was complexed with T-cell intracellular antigen-1 (TIA-1), a core SG component, and that the complex formation was correlated with Sam68 SG recruitment. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1 in response to oxidative stress and suggest that cytoplasmic SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions.     

Redox regulation of protein expression in Saccharomyces cerevisiae mitochondria: possible role of VDAC

Using Saccharomyces cerevisiae mutants depleted of either isoform of VDAC (voltage dependent anion selective channel) we studied the role of the cytosol and mitochondria redox states in regulation of the expression levels of some mitochondrial proteins. The studied proteins are MnSOD and subunits of the protein import machinery of the mitochondrial outer membrane, i.e. Tom70, Tom40 and Tob55 (Sam50). We have shown that both the cytosol and mitochondria redox states depend on the presence of a given VDAC isoform. The cytosol redox state is mediated by VDAC1, although VDAC2 has a quantitative effect, whereas the mitochondria redox state depends on the presence of both VDAC isoforms. Moreover, we have shown that the cytosol redox status but not the mitochondrial one is decisive for the expression levels of the studied mitochondrial proteins. Thus, expression levels of some mitochondrial proteins is influenced by VDAC and this regulatory process at least partially does not require its channel activity as VDAC2 does not form a channel. Thus, VDAC can be regarded as a participant of signaling pathways in S. cerevisiae cells.     

Along the trail from Fraction I protein to Rubisco (ribulose bisphosphate carboxylase-oxygenase)

This historical minireview deals with events leading to the eventual discovery of Rubisco (ribulose bisphosphate carboxylase-oxygenase). This abundant leaf protein is not only responsible for the net fixation of CO₂ in all plants, but also causes the loss of carbon through photorespiration. The latter is a special ‘problem’ of the so-called ‘C₃’ plants. The protein was first called ‘Fraction 1 protein’ before it was recognized to be the same as Rubisco. Instead of reinventing words, text as needed has been freely used from three earlier publications (Wildman and Kwanyuen 1978; Wildman 1992, 1998) This revised version was published online in June 2006 with corrections to the Cover Date.     

RNA结合蛋白Sam68及其功能

细胞通过基因表达调控来应对外界刺激,其中影响mRNA稳定性及翻译效率的转录后调控发挥重要作用。RNA结合蛋白(RNA binding proteins, RBPs)是介导转录后调控的重要分子,Sam68（SRC associated in mitosis of 68 kD）是集信号转导特性与RNA激活功能于一身的RNA结合蛋白,参与转录、可变剪接及核输出等mRNA 的代谢过程,且Sam68可通过信号通路参与细胞应答、细胞周期调控和疾病发生等。最新研究表明,Sam68可通过非编码RNAs(noncoding RNA, ncRNAs)参与表观遗传、转录与转录后调控。本文在介绍Sam68结构和转录后修饰的基础上,着重讨论Sam68在信号转导、可变剪接、ncRNAs代谢、疾病发生等方面的最新研究进展。