Comprehensive proteomic analysis of the human spliceosome

The precise excision of introns from pre-messenger RNA is performed by the spliceosome, a macromolecular machine containing five small nuclear RNAs and numerous proteins. Much has been learned about the protein components of the spliceosome from analysis of individual purified small nuclear ribonucleoproteins and salt-stable spliceosome 'core' particles. However, the complete set of proteins that constitutes intact functional spliceosomes has yet to be identified. Here we use maltose-binding protein affinity chromatography to isolate spliceosomes in highly purified and functional form. Using nanoscale microcapillary liquid chromatography tandem mass spectrometry, we identify approximately 145 distinct spliceosomal proteins, making the spliceosome the most complex cellular machine so far characterized. Our spliceosomes comprise all previously known splicing factors and 58 newly identified components. The spliceosome contains at least 30 proteins with known or putative roles in gene expression steps other than splicing. This complexity may be required not only for splicing multi-intronic metazoan pre-messenger RNAs, but also for mediating the extensive coupling between splicing and other steps in gene expression.     

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.     

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%.     

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。     

The conserved protein DCN-1/Dcn1p is required for cullin neddylation in C. elegans and S. cerevisiae

SCF-type E3 ubiquitin ligases are multi-protein complexes required for polyubiquitination and subsequent degradation of target proteins by the 26S proteasome. Cullins, together with the RING-finger protein Rbx1, form the catalytic core of the ligase, and recruit the substrate-recognition module. Cycles of covalent modification of cullins by the ubiquitin-like molecule Nedd8 (neddylation) and removal of Nedd8 by the COP9 signalosome (deneddylation) positively regulate E3 ligase activity. Here we report the identification and analysis of a widely conserved protein that is required for cullin neddylation in the nematode Caenorhabditis elegans and the yeast Saccharomyces cerevisiae. C. elegans DCN-1 and S. cerevisiae Dcn1p (defective in cullin neddylation) are characterized by a novel UBA-like ubiquitin-binding domain and a DUF298 domain of unknown function. Consistent with their requirements for neddylation, DCN-1 and Dcn1p directly bind Nedd8 and physically associate with cullins in both species. Moreover, overexpression of Dcn1p in yeast results in the accumulation of Nedd8-modified cullin Cdc53p. Both in vivo and in vitro experiments indicate that Dcn1p does not inhibit deneddylation of Cdc53p by the COP9 signalosome, but greatly increases the kinetics of the neddylation reaction.     

The RNA splicing factor hSlu7 is required for correct 3' splice-site choice

The production of correctly spliced messenger RNA requires two catalytic splicing steps. During step II, exon 1 attacks an adenine-guanine (AG) dinucleotide at the 3' splice site. This AG is usually located between 18 and 40 nucleotides downstream from the branch site, and closer AGs are skipped in favour of AGs located more optimally downstream. At present, little is understood about how the correct AG is distinguished from other AGs. Here we describe a metazoan splicing factor (hSlu7) that is required for selection of the correct AG. In the absence of hSlu7, use of the correct AG is suppressed and incorrect AGs are activated. We investigated this loss of fidelity by analysing spliceosomes assembled in the absence of hSlu7. These studies reveal that exon 1 is loosely associated with these spliceosomes. Thus, the improperly held exon cannot access the correct AG, but can attack other AGs indiscriminately. We conclude that hSlu7 is required to hold exon 1 tightly within the spliceosome for attack on a prespecified AG.     

Conservation between yeast and man of a protein associated with U5 small nuclear ribonucleoprotein

The process of nuclear pre-messenger RNA splicing is similar in Saccharomyces cerevisiae and metazoan cells in that the two-step mechanism is identical and the reaction occurs in a large ribonucleoprotein complex, the spliceosome. Little is known, however, about the degree of conservation of splicing factors other than of the small nuclear RNAs (snRNAs). Yeast counterparts of the metazoan spliceosomal snRNAs (U1, U2, U4, U5 and U6) have been identified but, with the exception of U6, the yeast snRNAs are larger and sequence similarity is limited to short regions. By using antibodies against the yeast PRP8 protein, a pre-mRNA splicing factor of relative molecular mass 280,000 (Mr280K) stably associated with U5 small nuclear ribonucleoproteins (snRNPs), we have now identified an immunologically related protein in HeLa cell nuclear extracts. The HeLa cell protein has an Mr greater than 200K and is associated with purified 20S U5 snRNPs. This is the first report of phylogenetic conservation between yeast and man of a protein splicing factor.     

Isolation of an active step I spliceosome and composition of its RNP core

Formation of catalytically active RNA structures within the spliceosome requires the assistance of proteins. However, little is known about the number and nature of proteins needed to establish and maintain the spliceosome's active site. Here we affinity-purified human spliceosomal C complexes and show that they catalyse exon ligation in the absence of added factors. Comparisons of the composition of the precatalytic versus the catalytic spliceosome revealed a marked exchange of proteins during the transition from the B to the C complex, with apparent stabilization of Prp19-CDC5 complex proteins and destabilization of SF3a/b proteins. Disruption of purified C complexes led to the isolation of a salt-stable ribonucleoprotein (RNP) core that contained both splicing intermediates and U2, U5 and U6 small nuclear RNA plus predominantly U5 and human Prp19-CDC5 proteins and Prp19-related factors. Our data provide insights into the spliceosome's catalytic RNP domain and indicate a central role for the aforementioned proteins in sustaining its catalytically active structure.     

Dephosphorylated SRp38 acts as a splicing repressor in response to heat shock

The cellular response to stresses such as heat shock involves changes in gene expression. It is well known that the splicing of messenger RNA precursors is generally repressed on heat shock, but the factors responsible have not been identified. SRp38 is an SR protein splicing factor that functions as a general repressor of splicing. It is activated by dephosphorylation and required for splicing repression in M-phase cells. Here we show that SRp38 is also dephosphorylated on heat shock and that this dephosphorylation correlates with splicing inhibition. Notably, depletion of SRp38 from heat-shocked cell extracts derepresses splicing, and adding back dephosphorylated SRp38 specifically restores inhibition. We further show that dephosphorylated SRp38 interacts with a U1 small nuclear ribonucleoprotein particle (snRNP) protein, and that this interaction interferes with 5'-splice-site recognition by the U1 snRNP. Finally, SRp38-deficient DT40 cells show an altered cell-cycle profile consistent with a mitotic defect; they are also temperature sensitive and defective in recovery after heat shock. SRp38 thus plays a crucial role in cell survival under stress conditions by inhibiting the splicing machinery.     

A suppressor of a yeast splicing mutation (prp8-1) encodes a putative ATP-dependent RNA helicase

Five small nuclear RNAs (snRNAs) are required for nuclear pre-messenger RNA splicing: U1, U2, U4, U5 and U6. The yeast U1 and U2 snRNAs base-pair to the 5' splice site and branch-point sequences of introns respectively. The role of the U5 and U4/U6 small nuclear ribonucleoprotein particles (snRNPs) in splicing is not clear, though a catalytic role for the U6 snRNA has been proposed. Less is known about yeast splicing factors, but the availability of genetic techniques in Saccharomyces cerevisiae has led to the identification of mutants deficient in nuclear pre-mRNA splicing (prp2-prp27). Several PRP genes have now been cloned and their protein products characterized. The PRP8 protein is a component of the U5 snRNP and associates with the U4/U6 snRNAs/snRNP to form a multi-snRNP particle believed to be important for spliceosome assembly. We have isolated extragenic suppressors of the prp8-1 mutation of S. cerevisiae and present here the preliminary characterization of one of these suppressors, spp81. The predicted amino-acid sequence of the SPP81 protein shows extensive similarity to a recently identified family of proteins thought to possess ATP-dependent RNA helicase activity. The possible role of this putative helicase in nuclear pre-mRNA splicing is discussed.     

The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. Here we show that U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.     

Takagi-Sugeno模型在剪接位点识别中的应用

为降低基因剪接位点识别算法复杂度和计算量,根据剪接位点上下游序列的保守特性及碱基组成随位点邻近序列GC含量变化等统计特征,建立Takagi-Sugeno模糊模型.通过模型输出值和阈值比较,判断真实的剪接位点.基于模糊似然函数的模糊聚类算法确定模型结构和前件参数,并结合最小二乘法完成该模型后件参数的识别.仿真结果表明,该算法简单,可使模糊模型的结构辨识和参数辨识同时完成,从而实现模糊模型的快速识别;能够很好地提取剪接位点附近保守序列的统计特征,为剪接位点的识别提供一种新的方法.     

Fe65L2基因存在4种剪接形式

Ｆｅ６５Ｌ２是一种与老年痴呆症关蛋白－淀粉样肽前体蛋白相互作用的蛋白。报道了Ｆｅ６５Ｌ２基因的２种新的剪接形式,并证实了该基因存在４种剪接形式。这４种剪接形式是由同一个内含子中分别为６ｎｔ和２１ｎｔ的２段外显子序列按不同组合拼接而成的。     

Spliced leader RNAs from lower eukaryotes are trans-spliced in mammalian cells.

Exon sequences present on separate RNA molecules can be joined by trans-splicing in trypanosomatids, Euglena, and in the nematode and trematode worms. Trans-splicing involves an interaction between a 5' splice site present in a spliced leader RNA and a 3' splice site located near the 5' end of pre-messenger RNAs. In vitro trans-splicing of artificial mammalian pre-mRNAs has been reported, but the efficiency of splicing appears to depend on sequence complementarity between the two substrates. There has been speculation that some natural pre-mRNAs can be trans-spliced in mammalian cells in vivo, but alternative interpretations have not been ruled out. Here we show that spliced leader RNAs can be accurately trans-spliced in mammalian cells in vivo and in vitro. Both nematode and mammalian 3' splice sites can function as acceptors for trans-splicing in vivo. These results reveal functional conservation in the splicing machinery between lower eukaryotes and mammals, and they directly demonstrate the potential for trans-splicing in mammalian cells.     

Cleavage of pre-tRNAs by the splicing endonuclease requires a composite active site

Splicing is required for the removal of introns from a subset of transfer RNAs in all eukaryotic organisms. The first step of splicing, intron recognition and cleavage, is performed by the tRNA-splicing endonuclease, a tetrameric enzyme composed of the protein subunits Sen54, Sen2, Sen34 and Sen15. It has previously been demonstrated that the active sites for cleavage at the 5' and 3' splice sites of precursor tRNA are contained within Sen2 and Sen34, respectively. A recent structure of an archaeal endonuclease complexed with a bulge-helix-bulge RNA has led to the unexpected hypothesis that catalysis requires a critical 'cation-pi sandwich' composed of two arginine residues that serve to position the RNA substrate within the active site. This motif is derived from a cross-subunit interaction between the two catalytic subunits. Here we test the role of this interaction within the eukaryotic endonuclease and show that catalysis at the 5' splice site requires the conserved cation-pi sandwich derived from the Sen34 subunit in addition to the catalytic triad of Sen2. The catalysis of pre-tRNA by the eukaryotic tRNA-splicing endonuclease therefore requires a previously unrecognized composite active site.     

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.