Messenger RNA splicing in yeast: clues to why the spliceosome is a ribonucleoprotein

The removal of introns from eukaryotic messenger RNA precursors shares mechanistic characteristics with the self-splicing of certain introns, prompting speculation that the catalytic reactions of nuclear pre-messenger RNA splicing are fundamentally RNA-based. The participation of five small nuclear RNAs (snRNAs) in splicing is now well documented. Genetic analysis in yeast has revealed the requirement, in addition, for several dozen proteins. Some of these are tightly bound to snRNAs to form small nuclear ribonucleoproteins (snRNPs); such proteins may promote interactions between snRNAs or between an snRNA and the intron. Other, non-snRNP proteins appear to associate transiently with the spliceosome. Some of these factors, which include RNA-dependent adenosine triphosphatases, may promote the accurate recognition of introns.     

The Prp19p-associated complex in spliceosome activation

During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.     

Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1

Eukaryotic ribosomes are substantially larger and more complex than their bacterial counterparts. Although their core function is conserved, bacterial and eukaryotic protein synthesis differ considerably at the level of initiation. The eukaryotic small ribosomal subunit (40S) plays a central role in this process; it binds initiation factors that facilitate scanning of messenger RNAs and initiation of protein synthesis. We have determined the crystal structure of the Tetrahymena thermophila 40S ribosomal subunit in complex with eukaryotic initiation factor 1 (eIF1) at a resolution of 3.9 angstroms. The structure reveals the fold of the entire 18S ribosomal RNA and of all ribosomal proteins of the 40S subunit, and defines the interactions with eIF1. It provides insights into the eukaryotic-specific aspects of protein synthesis, including the function of eIF1 as well as signaling and regulation mediated by the ribosomal proteins RACK1 and rpS6e.     

Template boundary in a yeast telomerase specified by RNA structure

The telomerase ribonucleoprotein has a phylogenetically divergent RNA subunit, which contains a short template for telomeric DNA synthesis. To understand how telomerase RNA participates in mechanistic aspects of telomere synthesis, we studied a conserved secondary structure adjacent to the template. Disruption of this structure caused DNA synthesis to proceed beyond the normal template boundary, resulting in altered telomere sequences, telomere shortening, and cellular growth defects. Compensatory mutations restored normal telomerase function. Thus, the RNA structure, rather than its sequence, specifies the template boundary. This study reveals a specific function for an RNA structure in the enzymatic action of telomerase.     

番茄斑萎病毒属病毒S RNA序列比对

从生物信息数据库NCBI中搜索并下载最新有关番茄萎斑病毒属Tospovirus各种间S RNA片段全长序列以及该片段上NSs、N的核酸和蛋白质序列着手,运用DNAstar和DNAMAN以及NPSA等生物信息软件对其进行比对分析,结果发现,以NSs蛋白序列建立的系统进化树显示CCSV和TZSV之间的同源性为85.8%,IYSV和TYRV之间的同源性达90%,MYSV和PSMV间的同源性为97.5%;通过N蛋白序列分析显示,Tospovirus属中有7组同源性较高的种;在Tospovirus属病毒中非极性氨基酸含量最高,极性酸性氨基酸最少,比较TSWV中抗性破坏RB株系与普通株系在NSs、N核酸、蛋白质序列、氨基酸含量和蛋白质二级结构之间的差异,发现差异不显著.     

Plasma membrane compartmentalization in yeast by messenger RNA transport and a septin diffusion barrier

Asymmetric localization of proteins plays a key role in many cellular processes, including cell polarity and cell fate determination. Using DNA microarray analysis, we identified a plasma membrane protein-encoding mRNA (IST2) that is transported to the bud tip by an actomyosin-based process. mRNA localization created a higher concentration of IST2 protein in the bud compared with that of the mother cell, and this asymmetry was maintained by a septin-mediated membrane diffusion barrier at the mother-bud neck. These results indicate that yeast creates distinct plasma membrane compartments, as has been described in neurons and epithelial cells.     

Evidence for mating of the "asexual" yeast Candida albicans in a mammalian host

Since its classification nearly 80 years ago, the human pathogen Candida albicans has been designated as an asexual yeast. In this report, we describe the construction of C. albicans strains that were subtly altered at the mating-type-like (MTL) locus, a cluster of genes that resembles the mating-type loci of other fungi. These derivatives were capable of mating after inoculation into a mammalian host. C. albicans is a diploid organism, but most of the mating products isolated from a mouse host were tetrasomic for the two chromosomes that could be rigorously monitored and, overall, exhibited substantially higher than 2n DNA content. These observations demonstrated that C. albicans can recombine sexually.     

Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA

In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. Here, we present the crystal structure of a tetrameric exon junction core complex containing the DEAD-box adenosine triphosphatase (ATPase) eukaryotic initiation factor 4AIII (eIF4AIII) bound to an ATP analog, MAGOH, Y14, a fragment of MLN51, and a polyuracil mRNA mimic. eIF4AIII interacts with the phosphate-ribose backbone of six consecutive nucleotides and prevents part of the bound RNA from being double stranded. The MAGOH and Y14 subunits lock eIF4AIII in a prehydrolysis state, and activation of the ATPase probably requires only modest conformational changes in eIF4AIII motif I.     

Changing the acceptor identity of a transfer RNA by altering nucleotides in a "variable pocket"

The specificity of tRNA(Arg) (arginine transfer RNA) for aminoacylation (its acceptor identity) were first identified by computer analysis and then examined with amber suppressor tRNAs in Escherichia coli. On replacing two nucleotides in tRNA(Phe) (phenylalanine transfer RNA) with the corresponding nucleotides from tRNA(Arg), the acceptor identity of the resulting tRNA was changed to that of tRNA(Arg). The nucleotides used in the identity transformation occupy a "variable pocket" structure on the surface of the tRNA molecule where two single-stranded loop segments interact. The middle nucleotide in the anticodon also probably contributes to the interaction, since an amber suppressor of tRNA(Arg) had an acceptor identity for lysine as well as arginine.     

Antibody formation: initiation in "nonresponder" mice by macrophage synthetic polypeptide RNA

The RNA extracted from normal peritoneal macrophages exposed to a linear, random synthetic polypeptide, Glu(60)Ala(30)Tyr(10), initiated an immune response in C57B1/6J mice, although this strain responds very poorly to the antigen itself. From 10 to 150 micrograms of RNA obtained from mouse, rat, or rabbit macrophages was injected intraperitoneally into recipient mice, and specific antibody was detectable by passive hemagglutination 3 to 4 weeks later. Treatment of the RNA with ribonuclease destroyed its ability to initiate a specific immune response. The RNA contained by weight 0.02 percent of the (specific) antigen. The RNA obtained from cells incubated with a second polypeptide, Glu(36)Lys(24)Ala(40), initiated a response specific for this polymer. This RNA even when incubated in vitro with Glu(60)Ala(30)Tyr(10) failed to initiate antibody formation specific for Glu(60)Ala(30)Tyr(10).     

Structure of yeast poly(A) polymerase alone and in complex with 3'-dATP

Polyadenylate [poly(A)] polymerase (PAP) catalyzes the addition of a polyadenosine tail to almost all eukaryotic messenger RNAs (mRNAs). The crystal structure of the PAP from Saccharomyces cerevisiae (Pap1) has been solved to 2.6 angstroms, both alone and in complex with 3'-deoxyadenosine triphosphate (3'-dATP). Like other nucleic acid polymerases, Pap1 is composed of three domains that encircle the active site. The arrangement of these domains, however, is quite different from that seen in polymerases that use a template to select and position their incoming nucleotides. The first two domains are functionally analogous to polymerase palm and fingers domains. The third domain is attached to the fingers domain and is known to interact with the single-stranded RNA primer. In the nucleotide complex, two molecules of 3'-dATP are bound to Pap1. One occupies the position of the incoming base, prior to its addition to the mRNA chain. The other is believed to occupy the position of the 3' end of the mRNA primer.     

The cutaneous "rabbit": a perceptual illusion

Anomalous localizations of mechanical and electrical cutaneous pulses are produced when widely separated bodily points are successively stimulated with trains of taps. The observer experiences a manifold of discrete "phantom" impressions connecting the points actually touched. The theoretical basis for this perceptual phenomenon is not understood, but some boundary conditions are specified.     

Class II aminoacyl transfer RNA synthetases: crystal structure of yeast aspartyl-tRNA synthetase complexed with tRNA(Asp)

The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.     

Psilocybin: reaction with a fraction of rat brain

Psilocybin, a hallucinogen, formed a blue color with a subfraction of rat-brain mitochondria believed to contain nerve-ending particles. Color formation increased with pH, did not require oxygen, and involved a component that could not be solubilized. The effect was not shown by chemically related neuroactive compounds, such as bufotenine and serotonin, and was antagonized by only tyramine or ethylenediaminetetraacetic acid.