鱼类基因内含子研究进展

内含子是指断裂基因中的非编码区序列,在编码蛋白质前被去除。在高等生物中,内含子的长度远大于外显子,大部分随机突变会发生在内含子中。因此,内含子的存在使高等生物对突变的耐受能力大大增强了。研究表明,内含子可以提高基因表达效率;影响RNA的转录、剪接加工、出核孔以及翻译等过程;启动某些基因的表达;并通过选择性剪接调控基因的表达。内含子功能的研究成果给当前鱼类免疫基因研究开拓了全新的视野。对内含子的分类、剪接、功能以及鱼类内含子研究的新进展进行了综述,并展望了内含子在鱼类免疫基因研究中的应用。     

Bioinformatics analysis of alternative splicing

Over the past few years, the analysis of alternative splicing using bioinformatics has emerged as an important new field, and has significantly changed our view of genome function. One exciting front has been the analysis of microarray data to measure alternative splicing genome-wide. Pioneering studies of both human and mouse data have produced algorithms for discerning evidence of alternative splicing and clustering genes and samples by their alternative splicing patterns. Moreover, these data indicate the presence of alternative splice forms in up to 80 per cent of human genes. Comparative genomics studies in both mammals and insects have demonstrated that alternative splicing can in some cases be predicted directly from comparisons of genome sequences, based on heightened sequence conservation and exon length. Such studies have also provided new insights into the connection between alternative splicing and a variety of evolutionary processes such as Alu-based exonisation, exon creation and loss. A number of groups have used a combination of bioinformatics, comparative genomics and experimental validation to identify new motifs for splice regulatory factors, analyse the balance of factors that regulate alternative splicing, and propose a new mechanism for regulation based on the interaction of alternative splicing and nonsense-mediated decay. Bioinformatics studies of the functional impact of alternative splicing have revealed a wide range of regulatory mechanisms, from NAGNAG sites that add a single amino acid; to short peptide segments that can play surprisingly complex roles in switching protein conformation and function (as in the Piccolo C2A domain); to events that entirely remove a specific protein interaction domain or membrane anchoring domain. Common to many bioinformatics studies is a new emphasis on graph representations of alternative splicing structures, which have many advantages for analysis.     

鱼类低温应激反应的调控机制

鱼类遭受低温胁迫易产生分子、细胞和组织损伤, 甚至导致个体死亡。鱼类机体细胞感受到低温刺激后, 通过多种应激通路将低温信号传递至细胞核, 启动低温应激反应, 建立新的胞内稳态, 从而增强抗寒能力。低温可激活鱼类内分泌系统释放皮质醇和甲状腺素等激素, 调控代谢、渗透压和免疫反应, 最终引起生理和行为变化。鱼类低温应激反应受表观遗传修饰、转录和翻译、前体RNA可变剪接和蛋白质翻译后修饰等多层级的复杂调控。目前, 采用多组学技术已经鉴定到大量与低温响应相关的效应基因和调控通路。研究表明, 能量代谢和抗氧化应激反应在鱼类抗寒能力的建成起着重要作用。其他环境因子(如低氧和盐度)及鱼体生理状态(如饥饿和营养)也影响鱼类对低温刺激的反应和抗寒能力。鉴定抗寒相关的分子标记并解析其关联基因的作用机制对鱼类的抗寒育种具有重要意义。     

鱼类Toll样受体及其信号传导的研究进展

鱼类是脊椎动物中的一个重要类群, 在其生存与进化的过程中, 免疫系统担负着保护鱼类免受病原感染的重任, 其中Toll样受体家族等介导的先天性免疫是鱼类抗病免疫的第一道防线, 并在连接先天性免疫与获得性免疫反应中起着桥梁作用. 虽然从无脊椎动物到高等脊椎动物, Toll样受体家族内多数成员在蛋白质结构与功能上都较为保守, 但是鱼类作为最低等的脊椎动物, 在其进化过程中又形成了一些特有Toll样受体分子, 其剪接类型也更丰富; 鱼类Toll样受体家族介导的免疫识别、免疫信号传导、激活和调控方式与高等脊椎动物也不尽相同. 文章主要综述了鱼类Toll样受体的结构、种类、功能、多样性、免疫信号传导及其调控特点, 为深入了解鱼类的免疫反应奠定基础.       

Consequences of regulated pre-mRNA splicing in the immune system

Alternative splicing is widely recognized to be a ubiquitous and crucial mechanism for generating protein diversity and regulating protein expression. Numerous immunologically relevant genes have been found to undergo alternative splicing; however, there has been little effort to develop a coherent picture of how alternative splicing might be used as a general mechanism to regulate the function of the immune system. In this review, I summarize the mechanisms by which splicing is controlled in T cells, and discuss the role of alternative splicing and alternative isoform expression in the regulation of T-cell activation and function.     

黑曲霉木质纤维素降解酶基因的可变剪接分析及验证

黑曲霉Aspergillus niger因能够产生大量的木质纤维素降解酶而在木质纤维素资源利用中发挥重要作用。目前,有关黑曲霉基因组中与木质纤维素降解相关的基因是否存在可变剪接的情况尚不清楚。本研究以黑曲霉CBS513.88菌株为研究对象,采用rMATS和ABLas两种方法对黑曲霉在葡萄糖为唯一碳源(G组)和小麦秸秆为唯一碳源(WS组)下的56个木质纤维素降解酶基因的可变剪接事件进行分析,并通过RT-PCR扩增和内含子特异性扩增对3个典型基因的可变剪接体进行了验证。结果表明,ABLas可变剪接分析算法相较于rMATS分析算法更为准确,ABLas分析算法显示G组和WS组共有21个木质纤维素降解酶基因出现了可变剪接,可变剪接类型以内含子保留(IR)为主,占所有可变剪接事件的82.85%。另外,G组和WS组发生可变剪接的木质纤维素降解酶基因也有所不同：G组发生可变剪接的基因为13个,WS组发生可变剪接的基因为14个,两组都发生可变剪接的基因为6个,这表明黑曲霉木质纤维素降解酶基因的可变剪接在不同生长条件下存在差异,另一方面,黑曲霉中众多可变剪接体的存在也为开发新型的木质纤维素降解酶资源提供基础。     

Identification of cells deficient in signaling-induced alternative splicing by use of somatic cell genetics

In recent years, a growing number of mammalian genes have been shown to undergo alternative splicing in response to extracellular stimuli. However, the factors and pathways involved in such signal-induced alternative splicing are almost entirely unknown. Here we describe a novel method for identifying candidate trans-acting factors that are involved in regulating mammalian alternative splicing, using the activation-induced alternative splicing of the human CD45 gene in T cells as a model system. We generated a cell line that stably expresses a CD45 minigene-based GFP reporter construct, such that the levels of green-fluorescent protein (GFP) expressed in the cell reflect the splicing state of the endogenous CD45 gene. Following mutagenesis of this cell line, and multiple rounds of selection for cells that displayed aberrant levels of GFP expression, we isolated several cell lines that are at least partially defective in their ability to support regulated alternative splicing of endogenous CD45 pre-mRNA in response to cell stimulation. Thus we have successfully isolated mutants in a mammalian alternative splicing pathway through use of a somatic cell-based genetic screen. This study clearly demonstrates the feasibility of using genetic screens to further our understanding of the regulation of mammalian splicing, particularly as it occurs in response to environmental cues.     

选择性剪接在低氧应答中的调控作用

细胞为适应低氧环境,其相关基因的表达方式发生了改变,其中选择性剪接在低氧应答调控过程中起到了重要的作用。低氧诱导因子介导的低氧应答信号通路在机体适应低氧环境过程中起到了十分重要的作用,低氧诱导因子剪接体通过此通路调控红细胞生成、血管生成、糖酵解等过程。而抑制性PAS蛋白质、脯氨酸羟化酶、促血管生长因子、芳香羟受体核转运蛋白剪接体则通过其它通路进行调控。选择性剪接不仅在低氧应答中起重要作用,而且与阿尔茨海默病、动脉粥样硬化、癌症等常见人类疾病相关。     

Alternative splicing and expression profile analysis of expressed sequence tags in domestic pig

Domestic pig （Sus scrofa domestica） is one of the most important mammals to humans. Alternative splicing is a cellular mechanism in eukaryotes that greatly increases the diversity of gene products. Expression sequence tags （ESTs） have been widely used for gene discovery, expression profile analysis, and alternative splicing detection. In this study, a total of 712,905 ESTs extracted from 101 different nonnormalized EST libraries of the domestic pig were analyzed. These EST libraries cover the nervous system, digestive system, immune system, and meat production related tissues from embryo, newborn, and adult pigs, making contributions to the analysis of alternative splicing variants as well as expression profiles in various stages of tissues. A modified approach was designed to cluster and assemble large EST datasets, aiming to detect alternative splicing together with EST abundance of each splicing variant. Much efforts were made to classify alternative splicing into different types and apply different filters to each type to get more reliable results. Finally, a total of 1,223 genes with average 2.8 splicing variants were detected among 16,540 unique genes. The overview of expression profiles would change when we take alternative splicing into account.