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1.
In metazoans, spliceosome assembly is initiated through recognition of the 5' splice site by U1 snRNP and the polypyrimidine tract by the U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF. U2AF is a heterodimer comprising a large subunit, U2AF65, and a small subunit, U2AF35. U2AF65 directly contacts the polypyrimidine tract and is required for splicing in vitro. In comparison, the role of U2AF35 has been puzzling: U2AF35 is highly conserved and is required for viability, but can be dispensed with for splicing in vitro. Here we use site-specific crosslinking to show that very early during spliceosome assembly U2AF35 directly contacts the 3' splice site. Mutational analysis and in vitro genetic selection indicate that U2AF35 has a sequence-specific RNA-binding activity that recognizes the 3'-splice-site consensus, AG/G. We show that for introns with weak polypyrimidine tracts, the U2AF35-3'-splice-site interaction is critical for U2AF binding and splicing. Our results demonstrate a new biochemical activity of U2AF35, identify the factor that initially recognizes the 3' splice site, and explain why the AG dinucleotide is required for the first step of splicing for some but not all introns. 相似文献
2.
Mackereth CD Madl T Bonnal S Simon B Zanier K Gasch A Rybin V Valcárcel J Sattler M 《Nature》2011,475(7356):408-411
Many cellular functions involve multi-domain proteins, which are composed of structurally independent modules connected by flexible linkers. Although it is often well understood how a given domain recognizes a cognate oligonucleotide or peptide motif, the dynamic interaction of multiple domains in the recognition of these ligands remains to be characterized. Here we have studied the molecular mechanisms of the recognition of the 3'-splice-site-associated polypyrimidine tract RNA by the large subunit of the human U2 snRNP auxiliary factor (U2AF65) as a key early step in pre-mRNA splicing. We show that the tandem RNA recognition motif domains of U2AF65 adopt two remarkably distinct domain arrangements in the absence or presence of a strong (that is, high affinity) polypyrimidine tract. Recognition of sequence variations in the polypyrimidine tract RNA involves a population shift between these closed and open conformations. The equilibrium between the two conformations functions as a molecular rheostat that quantitatively correlates the natural variations in polypyrimidine tract nucleotide composition, length and functional strength to the efficiency to recruit U2 snRNP to the intron during spliceosome assembly. Mutations that shift the conformational equilibrium without directly affecting RNA binding modulate splicing activity accordingly. Similar mechanisms of cooperative multi-domain conformational selection may operate more generally in the recognition of degenerate nucleotide or amino acid motifs by multi-domain proteins. 相似文献
3.
Genetic evidence for base pairing between U2 and U6 snRNA in mammalian mRNA splicing. 总被引:40,自引:0,他引:40
Removal of introns from eukaryotic nuclear messenger RNA precursors is catalysed by a large ribonucleoprotein complex called the spliceosome, which consists of four small nuclear ribonucleoprotein particles (U1, U2, U5, and U4/U6 snRNPs) and auxiliary protein factors. We have begun a genetic analysis of mammalian U2 snRNA by making second-site mutations in a suppressor U2 snRNA. Here we find that several mutations in the 5' end of U2 (nucleotides 3-8) are deleterious and that one of these can be rescued by compensatory base changes in the 3' end of U6 (nucleotides 92-95). The results demonstrate genetically that the base-pairing interaction between U2 (nucleotides 3-11) and U6 snRNA (nucleotides 87-95), originally proposed on the basis of psoralen photocrosslinking experiments, can influence the efficiency of mRNA splicing in mammals. The U2/U6 interaction in yeast, however, is fairly tolerant to mutation (D.J. Field and J.D. Friesen, personal communication), emphasizing the potential for facultative RNA interactions within the spliceosome. 相似文献
4.
Base pairing between U2 and U6 snRNAs is necessary for splicing of a mammalian pre-mRNA. 总被引:44,自引:0,他引:44
Splicing of pre-messenger RNA in eukaryotic cells occurs in a multicomponent complex termed the spliceosome, which contains small nuclear ribonucleoprotein particles (snRNPs), protein factors and substrate pre-mRNA. Assembly of the spliceosome involves the stepwise binding of snRNPs and protein factors to the pre-mRNA through a poorly understood mechanism which probably involves specific RNA-RNA, RNA-protein and protein-protein interactions. Of particular interest are the interactions between snRNPs, which are likely to be important not only for assembly of the spliceosome but also for catalysis. U1 snRNP interacts with the 5' splice site and U2 snRNP with the branch site of the pre-mRNA; both of these interactions involve Watson-Crick base pairing. But very little is known about how other factors such as the U4/U6 and U5 snRNPs reach the spliceosome and function in splicing. Here we report evidence that U6 snRNA interacts directly with U2 snRNA by a mechanism involving base-pairing, and that this interaction can be necessary for splicing of a mammalian pre-mRNA in vivo. 相似文献
5.
6.
Cloning of decay-accelerating factor suggests novel use of splicing to generate two proteins 总被引:9,自引:0,他引:9
Decay-accelerating factor (DAF), a glycoprotein that is anchored to the cell membrane by phosphatidylinositol, binds activated complement fragments C3b and C4b, thereby inhibiting amplification of the complement cascade on host cell membranes. Here, we report the molecular cloning of human DAF from HeLa cells. Analysis of DAF complementary DNAs revealed two classes of DAF messenger RNA, one apparently derived from the other by a splicing event that causes a coding frameshift near the C terminus. The apparent 'intron' sequence contains an Alu family member and encodes contiguous protein sequence. Two DAF proteins are therefore possible, having divergent C-terminal domains which differ in their hydrophobicity. Both mRNAs are found on polysomes, suggesting that both are translated. We propose that the major (90%) spliced DAF mRNA encodes membrane-bound DAF whereas the minor (10%) unspliced DAF mRNA may encode secreted DAF and we present expression data supporting this. The deduced DAF sequence contains four repeating units homologous to a consensus repeat found in a recently described family of complement proteins. 相似文献
7.
Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG. 相似文献
8.
Crystal structure of the RNA-binding domain of the U1 small nuclear ribonucleoprotein A 总被引:100,自引:0,他引:100
The crystal structure of the RNA binding domain of the U1 small nuclear ribonucleoprotein A, which forms part of the ribonucleoprotein complex involved in the excision of introns, has been solved. It contains a four-stranded beta sheet and two alpha helices. The highly conserved segments designated RNP1 and RNP2 lie side by side on the middle two beta strands. U1 RNA binding studies of mutant proteins suggest that the RNA binds to the four-stranded beta sheet and to the flexible loops on one end. 相似文献
9.
Crystal structure of nerve growth factor in complex with the ligand-binding domain of the TrkA receptor. 总被引:7,自引:0,他引:7
Nerve growth factor (NGF) is involved in a variety of processes involving signalling, such as cell differentiation and survival, growth cessation and apoptosis of neurons. These events are mediated by NGF as a result of binding to its two cell-surface receptors, TrkA and p75. TrkA is a receptor with tyrosine kinase activity that forms a high-affinity binding site for NGF. Of the five domains comprising its extracellular portion, the immunoglobulin-like domain proximal to the membrane (TrkA-d5 domain) is necessary and sufficient for NGF binding. Here we present the crystal structure of human NGF in complex with human TrkA-d5 at 2.2 A resolution. The ligand-receptor interface consists of two patches of similar size. One patch involves the central beta-sheet that forms the core of the homodimeric NGF molecule and the loops at the carboxy-terminal pole of TrkA-d5. The second patch comprises the amino-terminal residues of NGF, which adopt a helical conformation upon complex formation, packing against the 'ABED' sheet of TrkA-d5. The structure is consistent with results from mutagenesis experiments for all neurotrophins, and indicates that the first patch may constitute a conserved binding motif for all family members, whereas the second patch is specific for the interaction between NGF and TrkA. 相似文献
10.
Human U2 snRNA can function in pre-mRNA splicing in yeast 总被引:12,自引:0,他引:12
The removal of introns from messenger RNA precursors requires five small nuclear RNAs (snRNAs), contained within ribonucleoprotein particles (snRNPs), which complex with the pre-mRNA and other associated factors to form the spliceosome. In both yeast and mammals, the U2 snRNA base pairs with sequences surrounding the site of lariat formation. Binding of U2 snRNP to the highly degenerate branchpoint sequence in mammalian introns is absolutely dependent on an auxiliary protein, U2AF, which recognizes a polypyrimidine stretch adjacent to the 3' splice site. The absence of this sequence motif in yeast introns has strengthened arguments that the two systems are fundamentally different. Deletion analyses of the yeast U2 gene have confirmed that the highly conserved 5' domain is essential, although the adjacent approximately 950 nucleotides can be deleted without any phenotypic consequence. A 3'-terminal domain of approximately 100 nucleotides is also required for wild-type growth rates; the highly conserved terminal loop within this domain (loop IV) may provide specific binding contacts for two U2-specific snRNP proteins. We have replaced the single copy yeast U2 (yU2) gene with human U2 (hU2), expecting that weak or no complementation would provide an assay for cloning additional splicing factors, such as U2AF. We report here that hU2 can complement the yeast deletion with surprising efficiency. The interactions governing spliceosome assembly and intron recognition are thus more conserved than previously suspected. Paradoxically, the conserved loop IV sequence is dispensable in yeast. 相似文献
11.
12.
Nir2蛋白具有多个结构域,在生物体中可能发挥多种生理功能。本实验用MAD软件对大肠杆菌BL21(plys)原核表达的Nir2蛋白碳端进行了晶体结构分析。结果显示,硒蛋氨酸取代蛋白晶体及未取代蛋白晶体同属空间群P212121,晶胞参数a=109.468 Å, b=120.621 Å, c=70.315 Å,分辨率分别为2.2 Å和2.7 Å。一个不对称单位含有三个分子,含水量为48%。Nir2蛋白碳端由C片层和N片层构成,其中C片层与钙ATPase的催化区域P相似,N片层为许多不相关的功能性蛋白所共有。研究证明二者之间的静电作用是影响Nir2蛋白碳端与Pyk2 FERM区域结合的主要因素。该晶体结构的分析结果为进一步研究Nir2蛋白碳端的潜在功能提供了结构基础。 相似文献
13.
Yuan Shao Sheng Xiao Wenjing Zhai Dong Li Yanxin Liu Shilian Liu Dexian Zheng 《科学通报(英文版)》2000,45(24):2263-2266
In order to understand why CD2 has a dual action of transduction of activation or apoptosis signals in T cells under different
experimental conditions, we employed a yeast two-hybrid system to look for a binding protein of the cytoplasmic domain of
CD2 which may be involved in this issue. A human T cell cDNA library was screened by a cDNA encoding the cytoplasmic domain
of CD2 (Thr211-Gln336). The specificity of protein-protein interaction was verified by co-immunoprecipitation. The binding
protein obtained, designated CD2cBP, was found to be homologous tov-fos transformation effector protein (Fte-1). As Fte-1 plays a role in cell transformation, growth, protein synthesis and protein-import
into mitochondria, this result suggests that CD2cBP may be putatively involved in CD2-mediated signaling. 相似文献
14.
Tyrosine phosphorylation of vav proto-oncogene product containing SH2 domain and transcription factor motifs. 总被引:30,自引:0,他引:30
B Margolis P Hu S Katzav W Li J M Oliver A Ullrich A Weiss J Schlessinger 《Nature》1992,356(6364):71-74
15.
G Waksman D Kominos S C Robertson N Pant D Baltimore R B Birge D Cowburn H Hanafusa B J Mayer M Overduin 《Nature》1992,358(6388):646-653
Three-dimensional structures of complexes of the SH2 domain of the v-src oncogene product with two phosphotyrosyl peptides have been determined by X-ray crystallography at resolutions of 1.5 and 2.0 A, respectively. A central antiparallel beta-sheet in the structure is flanked by two alpha-helices, with peptide binding mediated by the sheet, intervening loops and one of the helices. The specific recognition of phosphotyrosine involves amino-aromatic interactions between lysine and arginine side chains and the ring system in addition to hydrogen-bonding interactions with the phosphate. 相似文献
16.
In 1975, Hamberg et al. reported evidence for the existence of an unstable platelet-aggregating factor which they named thromboxane A2 (TXA2) and for which they proposed a novel bicyclic oxetane structure (1, below) based on the short half-life of the factor (t1/2 (37 degrees C) = 32 s at pH 7.4) and the isolation of degradation products related to thromboxane (TXB2) (2, below). As natural TXA2 has not yet been isolated and characterized as a pure compound, we have synthesized the proposed structure (1) from TXB2 and compared its biological properties with those of authentic, biologically generated material. Here we present evidence that synthetic material having structure (1) is indistinguishable from platelet-derived TXA2 in various biological assays and that the proposed structure (1) for TXA2 is correct. 相似文献
17.
Crystal structure of the anthrax lethal factor. 总被引:13,自引:0,他引:13
A D Pannifer T Y Wong R Schwarzenbacher M Renatus C Petosa J Bienkowska D B Lacy R J Collier S Park S H Leppla P Hanna R C Liddington 《Nature》2001,414(6860):229-233
Lethal factor (LF) is a protein (relative molecular mass 90,000) that is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. Here we describe the crystal structure of LF and its complex with the N terminus of MAPKK-2. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity. 相似文献
18.
The Alu domain of the mammalian signal recognition particle (SRP) comprises the heterodimer of proteins SRP9 and SRP14 bound to the 5' and 3' terminal sequences of SRP RNA. It retards the ribosomal elongation of signal-peptide-containing proteins before their engagement with the translocation machinery in the endoplasmic reticulum. Here we report two crystal structures of the heterodimer SRP9/14 bound either to the 5' domain or to a construct containing both 5' and 3' domains. We present a model of the complete Alu domain that is consistent with extensive biochemical data. SRP9/14 binds strongly to the conserved core of the 5' domain, which forms a U-turn connecting two helical stacks. Reversible docking of the more weakly bound 3' domain might be functionally important in the mechanism of translational regulation. The Alu domain structure is probably conserved in other cytoplasmic ribonucleoprotein particles and retroposition intermediates containing SRP9/14-bound RNAs transcribed from Alu repeats or related elements in genomic DNA. 相似文献
19.
Crystal structures of the membrane-binding C2 domain of human coagulation factor V 总被引:23,自引:0,他引:23
Macedo-Ribeiro S Bode W Huber R Quinn-Allen MA Kim SW Ortel TL Bourenkov GP Bartunik HD Stubbs MT Kane WH Fuentes-Prior P 《Nature》1999,402(6760):434-439
Rapid and controlled clot formation is achieved through sequential activation of circulating serine proteinase precursors on phosphatidylserine-rich procoagulant membranes of activated platelets and endothelial cells. The homologous complexes Xase and prothrombinase, each consisting of an active proteinase and a non-enzymatic cofactor, perform critical steps within this coagulation cascade. The activated cofactors VIIIa and Va, highly specific for their cognate proteinases, are each derived from precursors with the same A1-A2-B-A3-C1-C2 architecture. Membrane binding is mediated by the C2 domains of both cofactors. Here we report two crystal structures of the C2 domain of human factor Va. The conserved beta-barrel framework provides a scaffold for three protruding loops, one of which adopts markedly different conformations in the two crystal forms. We propose a mechanism of calcium-independent, stereospecific binding of factors Va and VIIIa to phospholipid membranes, on the basis of (1) immersion of hydrophobic residues at the apices of these loops in the apolar membrane core; (2) specific interactions with phosphatidylserine head groups in the groove enclosed by these loops; and (3) favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups. 相似文献
20.
Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing 总被引:13,自引:0,他引:13
U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP. 相似文献