SRrp37, a novel splicing regulator located in the nuclear speckles and nucleoli,interacts with SC35 and modulates alternative pre‐mRNA splicing in vivo

We report here the identification and characterization of a novel SR‐related protein, referred to as SRrp37, based on its apparent molecular weight and subcellular location. SRrp37 was identified through a yeast two‐hybrid screen during the course of searching for proteins interacting with pNO40, a ribosomal 60S core subunit. SRrp37 exhibited two alternative spliced isoforms generated by differential usage of the translation start site with the longer one, SRrp37, initiating at first exon and the shorter, SRrp37‐2, starting from exon 2. Three distinct motifs can be discerned in the SRrp37 protein: (1) a serine–arginine (SR) dipeptide enriched domain, (2) a polyserine stretch, and (3) a potential nucleolar localization signal comprising a long array of basic amino acids. SRrp37's message was translated in tissue‐specific patterns with both isoforms expressed at comparable levels in tissues showing expression. Indirect immunofluorescence analysis with an anti‐SRrp37 antibody, as well as an experiment using myc‐tagged proteins, demonstrated that SRrp37 was localized in nucleoli and nuclear speckles. GST pull‐down assay showed that SRrp37 interacted physically with SC35. Using adenovirus E1A and chimeric calcitonin/dhfr constructs as splicing reporter minigenes, we found that SRrp37 modulated alternative 5′ and 3′ splicing in vivo. Together, SRrp37 may participate directly in splicing regulation or indirectly through interaction with SC35. Studies on this novel splicing regulator may provide new information on the intricate splicing machinery as related to the RNA metabolism involving processing of mRNA and rRNA. J. Cell. Biochem. 108: 304–314, 2009. © 2009 Wiley‐Liss, Inc.     

Simulations of Tubulin Sheet Polymers as Possible Structural Intermediates in Microtubule Assembly

The microtubule assembly process has been extensively studied, but the underlying molecular mechanism remains poorly understood. The structure of an artificially generated sheet polymer that alternates two types of lateral contacts and that directly converts into microtubules, has been proposed to correspond to the intermediate sheet structure observed during microtubule assembly. We have studied the self-assembly process of GMPCPP tubulins into sheet and microtubule structures using thermodynamic analysis and stochastic simulations. With the novel assumptions that tubulins can laterally interact in two different forms, and allosterically affect neighboring lateral interactions, we can explain existing experimental observations. At low temperature, the allosteric effect results in the observed sheet structure with alternating lateral interactions as the thermodynamically most stable form. At normal microtubule assembly temperature, our work indicates that a class of sheet structures resembling those observed at low temperature is transiently trapped as an intermediate during the assembly process. This work may shed light on the tubulin molecular interactions, and the role of sheet formation during microtubule assembly.     

Identification of the microbial diversity of wastewater nutrient removal processes using molecular biotechnology

The microbial communities of three sludge samples were identified by using a combined cloning-Denaturing Gradient Gel Electrophoresis (DGGE) method. Two communities were taken from the aerobic and the rotating biological contactor (RBC) of a novel hybrid system, named TNCU-I, which is a combined activated sludge-RBC bioprocess. The other sample was taken from the aerobic tank of a typical anaerobic-anoxic-aerobic (A2O) process. Acidovorax defluvii, Hydrogenophaga palleronii and Streptococcus suis were the most predominant bacteria, respectively, in TNCU-I activated sludge, TNCU-I RBC biofilm and A2O activated sludge, with abundances of 13.2%, 18.7% and 16.5%. Other predominant bacteria and their characteristics in wastewater treatment process are also described.     

Rapid Layer‐Specific Annealing Enabled by Ultraviolet LED with Estimation of Crystallization Energy for High‐Performance Perovskite Solar Cells

A rapid layer‐specific annealing on perovskite active layer enabled by ultraviolet (UV) light‐emitting diode (LED) is demonstrated and efficiency close to 19% is achieved in a simple planar inverted structure ITO/PEDOT:PSS/MAPbI₃/PC₇₁BM/Al without any device engineering. These results demonstrate that if the UV dosage is well managed, UV light is capable of annealing perovskite into high‐quality film rather than simply damaging it. Different in principle from other photonic treatment techniques that can heat up and damage underlying films, the UV‐LED‐annealing method enables layer‐specific annealing because LED light source is able to provide a specific UV wavelength for maximum light absorption of target film. Moreover, the layer‐specific photonic treatment allows accurate estimation of the crystallization energy required to form perovskite film at device quality level.     

Modulation of transcriptional corepressor activity of prospero-related homeobox protein (Prox1) by SUMO modification

Prospero-related homeobox protein (Prox1) plays essential roles in the development of many tissues and organs. In the present study, we show that Prox1 is modified by the small ubiquitin-like protein SUMO-1 in cultured cells. Mutation analysis identified at least four potential sumoylation sites within the repression domain of Prox1. Our data indicate that sumoylation of Prox1 reduces its interaction with HDAC3 and as a result downregulates its corepressor activity. These findings suggest that sumoylation may serve as a novel mechanism for the regulation of Prox1’s corepressor activity.

Structured summary

MINT-6787569:

PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with HDAC3 (uniprotkb:O15379) by anti tag coimmunoprecipitation (MI:0007)

MINT-6787767:

PROX1 (uniprotkb:Q92786) physically interacts (MI:0218) with SUMO-1 (uniprotkb:P63165) by anti tag coimmunoprecipitation (MI:0007)

     

烟曲霉蛋白质分泌载体的构建及酸性磷酸酯酶的细胞定位

[目的]在酵母细胞中蛋白质的糖基磷酸肌醇化(GPI)修饰是将GPI定位于细胞膜或细胞壁的信号.目前已对酵母GPI蛋白的细胞定位信号有一定了解,但对丝状真菌GPI蛋白的定位则了解甚少.AfPhoA是丝状真菌烟曲霉(Aspergillus fumigatus)的酸性磷酸酯酶,是GPI修饰的蛋白.该蛋白首先分离自细胞膜,随后又发现该蛋白与细胞壁结合.分析其C-端序列也未发现已知的定位信号,因此目前还不能确定其细胞定位.[方法]我们以绿色荧光蛋白(GFP)作为报告分子,将AfPhoA的C-端序列与GFP的C-端融合后检测融合GFP的细胞定位.[结果]我们用烟曲霉几丁质酶AfChiB1的启动子和N-端信号肽构建了可在烟曲霉中分泌表达GFP的表达载体pchiGFP.在此基础上将AfPhoA的C-端与GFP融合,融合质粒与pCDA14共转化烟曲霉后筛选到一株转化子.该转化子可表达融合GFP,在诱导和非诱导条件下,融合GFP均主要分布在细胞膜上,随培养时间的延长,融合GFP在细胞壁上也有少量分布;在培养上清液中只能检出约30KD的GFP融合蛋白,而没有完整的GFP融合蛋白,推测为从GPI锚上水解释放的.[结论]我们的研究结果表明,AfPhoA蛋白GPI修饰的作用是使该蛋白定位于细胞膜.本研究不仅初步确定了AfPhoA蛋白GPI修饰的细胞膜定位功能,而且为烟曲霉基因与蛋白质功能的研究建立了一个有效表达系统.     

新城疫不同毒株交叉鸡胚中和指数及其与F和HN基因变异的相关性

选取13株国内2001～2004年分离的新城疫流行病毒(Newcastle disease virus,NDV),经蚀斑纯化,克隆其融合蛋白(F)和血凝素.神经氨酸酶(HN)基因,结合疫苗株La Sota、Clone30和国内标准强毒株F48E9等的基因序列,进行遗传变异分析.利用纯化的病毒制备特异阳性血清,进行鸡胚交叉中和试验,确定不同NDV毒株之间的抗原相关性,并与NDV不同毒株之间的HN和F基因核苷酸(氨基酸)同源性进行相关比较.结果表明:病毒中和指数与HN基因的核苷酸(氨基酸)同源性显著相关(P<0.01,r=-0.35),与F基因呈弱相关(P<0.05,r=0.20),而与F基因前374bp的核甘酸同源性不相关.这表明,NDV的分子变异已经对NDV的抗原性变异产生了影响,研制新型的疫苗成为必然.