Inhibition of In Vitro Splicing of a Group I Intron of Pneumocystis carinii

Unlike its mammalian hosts, the opportunistic fungal pathogen Pneumocystis carinii harbors group I self-splicing introns in its chromosomal genes encoding rRNA. This difference between pathogen and host suggests that intron splicing is a promising target for chemotherapy. We have found that intron splicing in vitro is inhibited by the anti- Pneumocystis agent pentamidine and by a series of pentamidine analogues, as well as by some aminoglycosides, tetracycline, L-arginine and ethidium bromide. Further studies will be needed to determine if this is the mechanism of action of pentamidine against P. carinii .     

The Peperomia Mitochondrial coxI Group I Intron: Timing of Horizontal Transfer and Subsequent Evolution of the Intron

The Peperomia polybotrya coxI gene intron is the only currently reported group I intron in a vascular plant mitochondrial genome and it likely originated by horizontal transfer from a fungal donor. We provide a clearer picture of the horizontal transfer and a portrayal of the evolution of the group I intron since it was gained by the Peperomia mitochondrial genome. The intron was transferred recently in terms of plant evolution, being restricted to the single genus Peperomia among the order Piperales. Additional support is presented for the suggestion that a recombination/repair mechanism was used by the intron for integration into the Peperomia mitochondrial genome, as a perfect 1:1 correspondence exists between the intron's presence in a species and the presence of divergent nucleotide markers flanking the intron insertion site. Sequencing of coxI introns from additional Peperomia species revealed that several mutations have occurred in the intron since the horizontal transfer, but sequence alterations have not caused frameshifts or created stop codons in the intronic open reading frame. In addition, two coxI pseudogenes in Peperomia cubensis were discovered that lack a large region of coxI exon 2 and contain a truncated version of the group I intron that likely cannot be spliced out. Received: 29 May 1997 / Accepted: 1 November 1997     

糯性水稻中蜡质基因第1内含子的剪接活性

以前的研究结果表明 ,非糯性的稻米中直链淀粉的含量与各品种中蜡质基因 (Wx)第 1内含子被剪接的效率有关 ,即剪接效率高的品种直链淀粉含量也高。为弄清糯米中不含直链淀粉是否也与此内含子剪接效率有关系 ,构建了蜡质基因启动子 (来自籼稻品种2 32 )、蜡质基因第 1内含子 [来自籼稻品种 2 32 (高直链淀粉含量的品种 )或粳稻品种寒丰 6 36 6 (中、低直链淀粉含量的品种 ) ]与GUS报告基因组成的两种嵌合基因 ,将含有这两种嵌合基因的质粒分别转化进粳性糯稻品种奉糯 5 93中 ,同时还分别转化进非糯性的籼稻品种特青和粳稻品种中花 11中作为对照 ,测定它们的抗性愈伤组织与转基因植株未成熟种子胚乳中的GUS酶活性。结果表明与在非糯性的籼稻和粳稻中一样 ,这两种嵌合质粒在不合成直链淀粉的糯稻中也有相当水平的表达。从此结果可以推测 ,糯稻品种有从嵌合基因的转录本中剪接蜡质基因第 1内含子的正常能力 ,而在糯稻中缺乏直链淀粉可能是糯稻蜡质基因第1内含子中的其它突变所造成的     

Phylogeny and Self-Splicing Ability of the Plastid tRNA-Leu Group I Intron

Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7–3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present-day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants or a subset of the Stramenopiles (xanthophyceae/phaeophyceae) and frequent loss among green algae, as well as in the red algae and their secondary plastid derivatives (except the xanthophyceae/phaeophyceae lineage). Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host dependence likely evolved once in the common ancestor of land plants. In all other plastid lineages, these ribozymes could either self-splice or complete only the first step of autocatalysis. The first two authors (Dawn Simon and David Fewer) have contributed equally to this work. Present address (David Fewer): Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56, Viikinkaari 9, 00014 University of Helsinki, Helsinki, Finland     

Group I Introns from Zygomycota: Evolutionary Implications for the Fungal IC1 Intron Subgroup

The origins of fungal group I introns within nuclear small-subunit (nSSU) rDNA are enigmatic. This is partly because they have never been reported in basal fungal phyla (Zygomycota and Chytridiomycota), which are hypothesized to be ancestral to derived phyla (Ascomycota and Basidiomycota). Here we report group I introns from the nSSU rDNA of two zygomycete fungi, Zoophagus insidians (Zoopagales) and Coemansia mojavensis (Kickxellales). Secondary structure analyses predicted that both introns belong to the IC1 subgroup and that they are distantly related to each other, which is also suggested by different insertion sites. Molecular phylogenetic analyses indicated that the IC1 intron of Z. insidians is closely related to the IC1 intron inserted in the LSU rDNA of the basidiomycete fungus Clavicorona taxophila, which strongly suggests interphylum horizontal transfer. The IC1 intron of C. mojavensis has a low phylogenetic affinity to other fungal IC1 introns inserted into site 943 of nSSU rDNA (relative to E. coli 16S rDNA). It is noteworthy that this intron contains a putative ORF containing a His–Cys box motif in the antisense strand, a hallmark for nuclear-encoded homing endonucleases. Overall, molecular phylogenetic analyses do not support the placement of these two introns in basal fungal IC1 intron lineages. This result leads to the suggestion that fungal IC1 introns might have invaded or been transferred laterally after the divergence of the four major fungal phyla. Received: 8 February 2001 / Accepted: 1 November 2001     

A Tyrosyl-tRNA Synthetase Suppresses Structural Defects in the Two Major Helical Domains of the Group I Intron Catalytic Core

TheNeurospora crassamitochondrial tyrosyl-tRNA synthetase, the CYT-18 protein, functions in splicing group I introns by promoting the formation of the catalytically active structure of the intron RNA. The group I intron catalytic core is thought to consist of two extended helical domains, one formed by coaxial stacking of P5, P4, P6, and P6a (P4-P6 domain) and the other consisting of P8, P3, P7, and P9 (P3-P9 domain). To investigate how CYT-18 stabilizes the active RNA structure, we used anEscherichia coligenetic assay based on the phage T4tdintron to systematically test the ability of CYT-18 to compensate for structural defects in three key regions of the catalytic core: J3/4 and J6/7, connecting regions that form parts of the triple-helical-scaffold structure with the P4-P6 domain, and P7, a long- range base-pairing interaction that forms the guanosine-binding site and is part of the P3-P9 domain. Our results show that CYT-18 can suppress numerous mutations that disrupt the J3/4 and J6/7 nucleotide-triple interactions, as well as mutations that disrupt base-pairing in P7. CYT-18 suppressed mutations of phylogenetically conserved nucleotide residues at all positions tested, except for the universally conserved G-residue at the guanosine-binding site. Structure mapping experiments with selected mutant introns showed that the CYT-18-suppressible J3/4 mutations primarily impaired folding of the P4-P6 domain, while the J6/7 mutations impaired folding of both the P4-P6 and P3-P9 domains to various degrees. The P7 mutations impaired the formation of both P7 and P3, thereby grossly disrupting the P3-P9 domain. The finding that the P7 mutations also impaired formation of P3 provides evidence that the formation of these two long-range pairings is interdependent in thetdintron. Considered together with previous work, the nature of mutations suppressed by CYT-18 supports a model in which CYT-18 helps assemble the P4-P6 domain and then stabilizes the two major helical domains of the catalytic core in the correct relative orientation to form the intron's active site.     

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.     

HRP-2, the Caenorhabditis elegans Homolog of Mammalian Heterogeneous Nuclear Ribonucleoproteins Q and R, Is an Alternative Splicing Factor That Binds to UCUAUC Splicing Regulatory Elements

Alternative splicing is regulated by cis sequences in the pre-mRNA that serve as binding sites for trans-acting alternative splicing factors. In a previous study, we used bioinformatics and molecular biology to identify and confirm that the intronic hexamer sequence UCUAUC is a nematode alternative splicing regulatory element. In this study, we used RNA affinity chromatography to identify trans factors that bind to this sequence. HRP-2, the Caenorhabditis elegans homolog of human heterogeneous nuclear ribonucleoproteins Q and R, binds to UCUAUC in the context of unc-52 intronic regulatory sequences as well as to RNAs containing tandem repeats of this sequence. The three Us in the hexamer are the most important determinants of this binding specificity. We demonstrate, using RNA interference, that HRP-2 regulates the alternative splicing of two genes, unc-52 and lin-10, both of which have cassette exons flanked by an intronic UCUAUC motif. We propose that HRP-2 is a protein responsible for regulating alternative splicing through binding interactions with the UCUAUC sequence.Alternative pre-mRNA splicing is a mechanism for generating multiple mRNA isoforms from a single gene. This process can allow a gene to encode for more than one protein isoform. For some genes, it is a mechanism for regulating message stability through production of alternative mRNA isoforms that are substrates for the nonsense-mediated mRNA decay pathway (1). The majority of human genes undergo alternative splicing (2), and the process can be regulated in tissue-specific and developmental stage-specific manners. Current models propose that cis elements on the pre-mRNA, in exons and introns, serve as recognition sites for trans-acting protein factors that bind to the pre-mRNA and regulate assembly of the splicing machinery, thus regulating splice site choice (3).In recent years, a number of groups have employed bioinformatics techniques to identify cis splicing regulatory elements (4). These techniques include using multiple interspecies sequence alignments to identify conserved intronic regions, identification of short sequences in exons that are bounded by weak consensus splice sites, and identification of common intronic sequences flanking similarly regulated alternative exons (5–9). These efforts have added many new sequences to the list of known and potential splicing regulators. The identification of the protein factor partners for these sequences will be important for understanding their function in alternative splicing regulation.Experimental approaches have identified alternative splicing factors that interact with specific cis elements (10), but the number of trans factors discovered still lags behind the number of newly identified cis element partners. Some examples of well-characterized cis element/trans-acting factor interactions include the NOVA K homology domain splicing factor binding to the sequence UCAY (11), the FOX splicing factors binding to the sequence UGCAUG (12–14), and hnRNP³ F/H proteins binding to the sequence GGGG (15, 16). By using cross-linking immunoprecipitation followed by large scale sequencing, entire catalogs of RNAs that the splicing factors NOVA, SF2/ASF, and FOX2 bind to in vivo have been determined (17–19). These approaches have led to models for how the proteins binding to their cis regulatory elements may alter splicing. These models include a role for the relative position of a cis element to an alternative cassette exon in determining alternative exon inclusion or skipping (18, 19).In a previous bioinformatics analysis of evolutionarily conserved intronic sequences flanking alternatively spliced exons, we identified the hexamer sequence UCUAUC as a novel splicing regulatory element (8). UCUAUC is found flanking both sides of alternative exon 16 of the unc-52 gene of Caenorhabditis elegans. Genetic analysis of a class of viable unc-52 mutants led to the discovery that exons 16–18 are alternative cassette exons and that every combination of skipping and inclusion of these three exons occurs (20). This splicing is regulated by the alternative splicing factor MEC-8 (21). Fig. 1A shows a schematic diagram of the alternatively spliced region of unc-52, with the MEC-8-enhanced alternative splicing events indicated. Using an unc-52 splicing reporter trans gene containing alternative exons 15–19, we previously reported that alternative splicing is regulated by the intronic motif UCUAUC in the intron downstream of exon 16 (8). In addition we showed that this element works cooperatively with a UGCAUG hexamer (the consensus FOX-1-binding site) in the upstream intron to regulate alternative splicing (8).Open in a separate windowFIGURE 1.RNA affinity chromatography identifies HRP-2 as binding to UCUAUC elements. A, schematic representation of the alternatively spliced region of unc-52 (adapted from Ref. 21). The alternative splicing events promoted by MEC-8 are indicated by bold lines. The lines next to introns 15 and 16 are the sites of the UCUAUC elements in those introns whose sequences were used in the RNA affinity chromatography. B, table showing sequences of RNAs immobilized to beads in the RNA affinity chromatography experiment. C, Coomassie-stained SDS-PAGE analysis of RNA affinity chromatography. C. elegans embryo extract was incubated with the different immobilized RNA substrates listed on top of the gel. Proteins identified by mass spectrometry are listed to the right of the gel, with arrows pointing to coincident protein bands. D, the left panel shows the silver stain result for the RNA affinity chromatography experiment. Each lane represents a different immobilized substrate, as indicated above. The band corresponding to HRP-2 is indicated by an arrow. The right panel is an immunoblot of the same gel using anti-HRP-2 polyclonal antibody. E, anti-HRP-2 immunoblot of an RNA affinity chromatography experiment for the indicated substrates.In this study, we report the results of a biochemical identification of a protein factor from C. elegans that binds to the UCUAUC intronic splicing regulatory element. We transcribed different short RNA sequences containing the UCUAUC element in its native intronic context, or as part of a repeating unit, and immobilized these onto agarose beads. After passing embryo extracts across these beads, we found that the protein HRP-2, the C. elegans homolog of the mammalian hnRNP Q/R proteins, binds to this sequence with high affinity. By using RNAi to reduce the level of HRP-2 in worms, we observed changes in alternative splicing of unc-52 and lin-10, two genes that contain UCUAUC elements in introns flanking alternative exons. We propose that HRP-2 is an alternative splicing factor that works through the UCUAUC intronic elements to regulate alternative splicing.