基于序列信息理论预测线虫基因选择性剪切位点

基因的选择性剪切使得在DNA上一段相同的序列翻译成多个不同的蛋白质序列.选择性剪切的出现把剪切位点分为选择性供体位点、组成性供体位点、选择性受体位点和组成性受体位点.基于EBI中的线虫基因选择性剪切位点数据库,选取不同位点的单碱基频率和序列片段的三联体频数作为参数,利用位置权重矩阵和离散增量结合支持向量机,对选择性剪切位点进行了理论预测.对选择性供体位点和选择性受体位点的预测成功率分别为63.78%和72.63%,特异性分别为68.02%和83.96%.     

Role of the ubiquitin-like protein Hub1 in splice-site usage and alternative splicing

Alternative splicing of pre-messenger RNAs diversifies gene products in eukaryotes and is guided by factors that enable spliceosomes to recognize particular splice sites. Here we report that alternative splicing of Saccharomyces cerevisiae SRC1 pre-mRNA is promoted by the conserved ubiquitin-like protein Hub1. Structural and biochemical data show that Hub1 binds non-covalently to a conserved element termed HIND, which is present in the spliceosomal protein Snu66 in yeast and mammals, and Prp38 in plants. Hub1 binding mildly alters spliceosomal protein interactions and barely affects general splicing in S. cerevisiae. However, spliceosomes that lack Hub1, or are defective in Hub1-HIND interaction, cannot use certain non-canonical 5' splice sites and are defective in alternative SRC1 splicing. Hub1 confers alternative splicing not only when bound to HIND, but also when experimentally fused to Snu66, Prp38, or even the core splicing factor Prp8. Our study indicates a novel mechanism for splice site utilization that is guided by non-covalent modification of the spliceosome by an unconventional ubiquitin-like modifier.     

The VASE exon downregulates the neurite growth-promoting activity of NCAM 140.

Axonal growth, guidance and synapse formation are controlled by receptors on neuronal growth cones that can recognize positive and inhibitory cues in the local microenvironment. Four well characterized receptor systems are known that recognize the growth-promoting activities associated with the extracellular matrix and the membranes of cells such as astrocytes, muscle cells and Schwann cells; these are the integrins and the homophilically binding cell adhesion molecules neural-cell adhesion molecule (NCAM), N-cadherin and L1 (refs 5-12). Alternative splicing generates 20-30 isoforms of NCAM and these can also be differentially glycosylated. There are two sites where alternative splicing changes the extracellular structure of membrane-bound NCAM and one of these (the MSD1 region) does not obviously affect function. Here we report that the variable alternatively spliced exon (VASE) in immunoglobulin domain 4 downregulates the neurite outgrowth-promoting activity of NCAM. The high level of VASE expression in the adult central as compared with peripheral nervous system could contribute to the poor regenerative capacity of the former.     

Mechanism of recognition of the 5' splice site in self-splicing group I introns

Group I introns include many mitochondrial ribosomal RNA and messenger RNA introns and the nuclear rRNA introns of Tetrahymena and Physarum. The splicing of precursor RNAs containing these introns is a two-step reaction. Cleavage at the 5' splice site precedes cleavage at the 3' splice site, the latter cleavage being coupled with exon ligation. Following the first cleavage, the 5' exon must somehow be held in place for ligation. We have now tested the reactivity of two self-splicing group I RNAs, the Tetrahymena pre-rRNA and the intron 1 portion of the Neurospora mitochondrial cytochrome b (cob) pre-mRNA, in the intermolecular exon ligation reaction (splicing in trans) described by Inoue et al. The different sequence specificity of the reactions supports the idea that the nucleotides immediately upstream from the 5' splice site are base-paired to an internal, 5' exon-binding site, in agreement with RNA structure models proposed by Davies and co-workers and others. The internal binding site is proposed to be involved in the formation of a structure that specifies the 5' splice site and, following the first step of splicing, to hold the 5' exon in place for exon ligation.     

真核基因选择性剪接机理的初步研究

真核基因表达调控是生命科学研究的前沿、热点。RNA的选择性剪接在真核基因表达调控中起着十分重要的作用。该文从已有数据出发 ,对真核基因的剪接机制以及选择性剪接的机制进行了初步的研究。结果表明 ,在真核基因的剪接过程中 ,可能存在一种“粗定位—细定位”的过程 ,即在剪接过程中首先有一粗略的定位过程 ,根据序列的嘌呤、嘧啶浓度特征寻找出剪接位点的大致位置 ;然后在这一基础上 ,根据几个保守碱基所提供的信息找到准确的剪接位点。这一结果对于进一步研究真核基因的剪接机制 ,特别是选择性剪接发生的机理有很大的启发。     

Are vertebrate exons scanned during splice-site selection?

Pairwise recognition of splice sites as a result of a scanning mechanism is an attractive model to explain the coordination of vertebrate splicing. Such a mechanism would predict a polarity-of-site recognition in the scanned unit, but no evidence for a polarity gradient across introns has been found. We have suggested that the exon rather than the intron is the unit of recognition in vertebrates and that polyadenylation and splicing factors interact during recognition of 3'-terminal exons. Interaction is reflected in maximal rates of in vitro polyadenylation. If scanning across the exon is operating during this interaction, then insertion of a 5' splice site should depress polyadenylation. Here we report recognition in vitro and in vivo of a 5' splice site situated within a 3'-terminal exon, and a concomitant depression of polyadenylation and ultraviolet crosslinking of a polyadenylation factor. Decreased crosslinking was only found when the 3' and 5' splice sites were within 300 nucleotides of each other. These results are consistent with an exon scanning mechanism for splice-site selection.     

中华蜜蜂幼虫肠道响应球囊菌胁迫的可变剪切基因分析

基于前期已获得的中华蜜蜂(简称中蜂)幼虫肠道转录组数据,利用TopHat2软件在正常(AcCK)及球囊菌胁迫的中蜂幼虫肠道样品(AcT1、AcT2、AcT3)中共鉴定出发生于9124个基因的57327个可变剪切事件,其中以基因间(17.68%)、可变3′端剪切(15.32%)、外显子跨越(14.12%)和可变5′端剪切(12.81%)类型为主.Venn分析结果显示4个肠道样品的共有可变剪切基因数为8111个,特有可变剪切基因数分别为272、189和385个.GO分类结果显示共有可变剪切基因涉及47个条目,AcT1、AcT2、AcT3的特有可变剪切基因分别富集于24、20和34个条目.KEGG代谢通路富集分析结果显示,共有可变剪切基因富集在327个代谢通路,基因富集数最多的是RNA转运、内质网蛋白加工及核糖体;AcT1、AcT2、AcT3的特有可变剪切基因分别富集在22、46和83个代谢通路.结果揭示了可变剪切基因在宿主的胁迫响应过程中的重要作用.     

CD44v6和p16在宫颈上皮内瘤变及宫颈癌中的表达及意义

目的探讨CD44v6和p16表达产物在宫颈上皮瘤样病变及宫颈癌中表达的意义。方法对正常宫颈鳞状上皮、宫颈上皮内瘤变（CIN）和宫颈癌组织共95例,采用免疫组织化学SP法,对宫颈癌变过程中CD44v6和p16蛋白进行研究,将结果进行统计分析。结果CD44v6蛋白在CIN中的表述高于正常宫颈上皮（P〈0.01）,在宫颈癌中的表达也高于正常宫颈上皮（P〈0.01）,且高于CIN中的表达（P〈0.01）,p16蛋白在CIN中的表达高于正常宫颈上皮（P〈0.01）,在宫颈癌中的表达也高于正常宫颈上皮（P〈0.01）;CD44v6和p16两种蛋白在CIN和宫颈癌中的表达无明显差异。结论CD44v6和p16蛋白的表达与宫颈癌的发生有关,提示这两种蛋白有可能作为高危人群早期筛查的一种免疫组化指标。     

组织芯片分析CD44v6和PCNA在口腔鳞癌及癌前病变中的表达及意义

目的：研究CDOv6和PCNA的表达与口腔鳞癌的发生、发展的关系。方法：应用组织芯片技术,通过免疫组织化学SP法分别检测一张组织芯片上117例组织样本中CD44v6、PCNA蛋白表达情况。结果：CD44v6在正常口腔黏膜组织和癌前病变呈阳性表达;癌组织中CD44v6的表达明显,但其表达程度随肿瘤恶性程度的增高而降低,PCNA的表达程度随着增生细胞和癌分级增加而升高。结论：在正常口腔黏膜上皮、增生上皮以及鳞癌中这两种蛋白的表达之间呈负相关。应用组织芯片大规模高效检测临床组织样本是可行的,具有快速、方便、经济、准确的特点。     

Translational control of intron splicing in eukaryotes

Most eukaryotic genes are interrupted by non-coding introns that must be accurately removed from pre-messenger RNAs to produce translatable mRNAs. Splicing is guided locally by short conserved sequences, but genes typically contain many potential splice sites, and the mechanisms specifying the correct sites remain poorly understood. In most organisms, short introns recognized by the intron definition mechanism cannot be efficiently predicted solely on the basis of sequence motifs. In multicellular eukaryotes, long introns are recognized through exon definition and most genes produce multiple mRNA variants through alternative splicing. The nonsense-mediated mRNA decay (NMD) pathway may further shape the observed sets of variants by selectively degrading those containing premature termination codons, which are frequently produced in mammals. Here we show that the tiny introns of the ciliate Paramecium tetraurelia are under strong selective pressure to cause premature termination of mRNA translation in the event of intron retention, and that the same bias is observed among the short introns of plants, fungi and animals. By knocking down the two P. tetraurelia genes encoding UPF1, a protein that is crucial in NMD, we show that the intrinsic efficiency of splicing varies widely among introns and that NMD activity can significantly reduce the fraction of unspliced mRNAs. The results suggest that, independently of alternative splicing, species with large intron numbers universally rely on NMD to compensate for suboptimal splicing efficiency and accuracy.     

CD44v6、E-cadherin和ki-67蛋白质与腮腺多形性腺瘤的生物学行为相关性研究

为检测腮腺多形性腺瘤中CD44v6、E-cadherin和ki-67蛋白表达,以探讨其与腮腺多形性腺瘤生物学行为的相关性。应用免疫组织化学SP法染色检测腮腺多形性腺瘤组、多形性腺瘤恶变组和瘤旁非瘤组织组各组间CD44v6、Ecadherin和ki-67蛋白表达。结果显示,(1)CD44v6、E-cadherin和ki-67在腮腺多形性腺瘤组中的阳性表达率分别为100%、96%和73.8%;(2)E-cadherin和ki-67在腮腺多形性腺瘤组、多形性腺瘤恶变组和瘤周非瘤组织间的阳性表达率均有差异,其表达与腮腺多形性腺瘤的浸润转移有关;(3)CD44v6在腮腺多形性腺瘤组与瘤周非瘤组织组,多形性腺瘤恶变组与瘤周非瘤组织组中的表达有统计学差异。由此可知,E-cadherin和ki-67与腮腺多形性腺瘤的生物学行为有关,可作为新的肿瘤标志物,其表达对判断预后有一定的价值。     

CD3delta couples T-cell receptor signalling to ERK activation and thymocyte positive selection

Thymocytes from mice lacking the CD3delta chain of the T-cell receptor (TCR), unlike those of other CD3-deficient mice, progress from a CD4- CD8- double-negative to a CD4+ CD8+ double-positive stage. However, CD3delta-/- double-positive cells fail to undergo positive selection, by which double-positive cells differentiate into more mature thymocytes. Positive selection is also impaired in mice expressing inactive components of the Ras/mitogen activated protein (MAP) kinase signalling pathway. Here we show that CD3delta-/- thymocytes are defective in the induction of extracellular signal-regulated protein kinase (ERK) MAP kinases upon TCR engagement, whereas activation of other MAP kinases is unaffected. The requirement for CD3delta maps to its extracellular or transmembrane domains, or both, as expression of a tail-less CD3delta rescues both ERK activation and positive selection in CD3delta-/- mice. Furthermore, the defect correlates with severely impaired tyrosine phosphorylation of the linker protein LAT, and of the CD3zeta chain that is localized to membrane lipid rafts upon TCR engagement. Our data indicate that the blockade of positive selection of CD3delta-/- thymocytes may derive from defective tyrosine phosphorylation of CD3zeta in lipid rafts, resulting in impaired activation of the LAT/Ras/ERK pathway.