Genetic and physical interactions between factors involved in both cell cycle progression and pre-mRNA splicing in Saccharomyces cerevisiae.

The PRP17/CDC40 gene of Saccharomyces cerevisiae functions in two different cellular processes: pre-mRNA splicing and cell cycle progression. The Prp17/Cdc40 protein participates in the second step of the splicing reaction and, in addition, prp17/cdc40 mutant cells held at the restrictive temperature arrest in the G2 phase of the cell cycle. Here we describe the identification of nine genes that, when mutated, show synthetic lethality with the prp17/cdc40Delta allele. Six of these encode known splicing factors: Prp8p, Slu7p, Prp16p, Prp22p, Slt11p, and U2 snRNA. The other three, SYF1, SYF2, and SYF3, represent genes also involved in cell cycle progression and in pre-mRNA splicing. Syf1p and Syf3p are highly conserved proteins containing several copies of a repeated motif, which we term RTPR. This newly defined motif is shared by proteins involved in RNA processing and represents a subfamily of the known TPR (tetratricopeptide repeat) motif. Using two-hybrid interaction screens and biochemical analysis, we show that the SYF gene products interact with each other and with four other proteins: Isy1p, Cef1p, Prp22p, and Ntc20p. We discuss the role played by these proteins in splicing and cell cycle progression.

Extensive genetic interactions between PRP8 and PRP17/CDC40, two yeast genes involved in pre-mRNA splicing and cell cycle progression.

Biochemical and genetic experiments have shown that the PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that plays a role during the second catalytic step of the splicing reaction. It was found recently that PRP17 is identical to the cell division cycle CDC40 gene. cdc40 mutants arrest at the restrictive temperature after the completion of DNA replication. Although the PRP17/CDC40 gene product is essential only at elevated temperatures, splicing intermediates accumulate in prp17 mutants even at the permissive temperature. In this report we describe extensive genetic interactions between PRP17/CDC40 and the PRP8 gene. PRP8 encodes a highly conserved U5 snRNP protein required for spliceosome assembly and for both catalytic steps of the splicing reaction. We show that mutations in the PRP8 gene are able to suppress the temperature-sensitive growth phenotype and the splicing defect conferred by the absence of the Prp17 protein. In addition, these mutations are capable of suppressing certain alterations in the conserved PyAG trinucleotide at the 3' splice junction, as detected by an ACT1-CUP1 splicing reporter system. Moreover, other PRP8 alleles exhibit synthetic lethality with the absence of Prp17p and show a reduced ability to splice an intron bearing an altered 3' splice junction. On the basis of these findings, we propose a model for the mode of interaction between the Prp8 and Prp17 proteins during the second catalytic step of the splicing reaction.

[2.2]paracyclophane/dehydrobenzoannulene hybrids: transannular delocalization in open-circuited conjugated macrocycles.

Enhanced global delocalization throughout the "stepped" π-electron systems of the [2.2]paracyclophane/dehydrobenzoannulene (PC/DBA) hybrids 1 and 2 is strongly suggested by a comparison of their electronic absorption spectra with those of model compounds with complete and interrupted classical aromatic delocalization. A distinct bathochromic shift (for 1) and greater absorption intensity at higher wavelengths (for 1 and 2) is observed versus the corresponding model hydrocarbons.

Synthesis and structure of a new [6.6]metacyclophane with enediyne bridges.

[reaction: see text] Synthesis and structure of a novel [6.6]metacyclophane with enediyne bridges is reported.

Revision of the absolute configuration of the tricyclic sesquiterpene (+)-kelsoene by chemical correlation and enantiospecific total synthesis of its enantiomer.

The absolute configuration of the recently identified sesquiterpene (+)-kelsoene was revised by chemical correlation with (R)-(+)-pulegone; the correct structure is (1R,2S,5R,6R,7R,8S)-2,8-dimethyl-6-(1-methylethenyl)tricyclo[5.3.0.0 (2,5)]decane.

Regulated splicing of adenovirus type 5 E4 transcripts and regulated cytoplasmic accumulation of E4 mRNA.

The E4 gene of human type C adenoviruses has been shown previously to give rise to an array of mRNAs via differential splicing. In this study, the pattern of expression of these mRNAs during lytic infection was examined, and two distinct temporal classes were defined. mRNAs of the early class were distinguished from those of the late class by the presence, in the early class, of a sequence in the 3' half of the mRNA that was removed as an intron in the late class. A single mRNA of the late class was found to show a strong dependence on the presence of the 55-kDa protein from region E1b and the open reading frame 6 protein from region E4 for its normal cytoplasmic accumulation. One feature of this mRNA that distinguishes it from other E4 mRNAs expressed at late times is the retention within it of an intron from the 5' half of E4; it may therefore be recognized as incompletely spliced by the host cell and retained in the nucleus. It is proposed that the E1b 55-kDa/E4 open reading frame 6 protein complex facilitates accumulation of this mRNA by overcoming this retention mechanism.

Open reading frames 1 and 2 of adenovirus region E4 are conserved between human serotypes 2 and 5.

The E4 region of human adenovirus type 2 is predicted to encode seven proteins as judged from its nucleotide sequence and the pattern of differential splicing of its transcript. Two of the open reading frames (ORFs), ORF1 and ORF2, had been identified as being disrupted in the recently published sequence of the related serotype 5 virus. These ORFs were resequenced and found to be intact in the wt300 strain of adenovirus type 5.

Expression of adenovirus type 5 E4 Orf2 protein during lytic infection.

The human adenovirus type 5 E4 transcription unit has the potential to encode at least seven distinct polypeptides from reading frames accessed by differential splicing of a single primary transcript. Only some of these polypeptides have yet been detected during viral infection of cultured cells. Mutational inactivation of the reading frames whose products have not been described has no apparent effect on the growth of virus in standard cultured human cell lines, indicating that these proteins, if they exist, have only a subtle, non-essential role in the replication cycle. We have raised an antiserum to one of these undefined products, E4 Orf2, expressed in bacteria. Using this reagent, it was possible to show that Orf2 was expressed during the lytic cycle in HeLa cells, being a soluble cytoplasmic component appearing with early kinetics. No association of Orf2 protein with other infected cell components was detected.

A self-fertile species of Steinernema from Indonesia: further evidence of convergent evolution amongst entomopathogenic nematodes?

More than 20 species of the entomopathogenic nematode Steinernema have been described; to date, all reproduce exclusively by cross-fertilization of male and female individuals. Steinernema sp. strain T87 from Indonesia was found to consist largely of self-fertile hermaphrodites. Progeny were produced by morphological females both in insects (Galleria mellonella) and in hanging drops of insect haemolymph inoculated with a single infective juvenile. Sperm were present in the oviduct of unmated morphological females. Approximately 1% of infective juveniles developed into males, and males were also present in the second generation where they constituted 1-6% of the population. Under the same conditions the related species Steinernema longicaudum strain CB2B displayed typical steinernematid reproduction: cross-fertilization and a 1:1 sex ratio. It is argued that the development of hermaphroditism in Steinernema sp. T87 represents convergent evolution with Heterorhabditis, the other major genus of entomopathogenic nematode.

Functional analyses of interacting factors involved in both pre-mRNA splicing and cell cycle progression in Saccharomyces cerevisiae.

Through a genetic screen to search for factors that interact with Prp17/Cdc40p, a protein involved in both cell cycle progression and pre-mRNA splicing, we identify three novel factors, which we call Syf1p, Syf2p, and Syf3 (SYnthetic lethal with cdc Forty). Here we present evidence that all three proteins are spliceosome associated, that they associate weakly or transiently with U6 and U5 snRNAs, and that Syf1p and Syf3p (also known as Clf1p) are required for pre-mRNA splicing. In addition we show that depletion of Syf1p or Syf3p results in cell cycle arrest at the G2/M transition. Thus, like Prp17/Cdc40p, Syf1p and Syf3p are involved in two distinct cellular processes. We discuss the likelihood that Syf1p, Syf2p, and Syf3p are components of a protein complex that assembles into spliceosomes and also regulates cell cycle progression.

The identification and characterization of a novel splicing protein, Isy1p, of Saccharomyces cerevisiae.

We have identified a novel splicing factor, Isy1p, through two-hybrid screens for interacting proteins involved in nuclear pre-mRNA splicing. Isy1p was tagged and demonstrated to be part of the splicing machinery, associated with spliceosomes throughout the splicing reactions. At least a portion of the Isy1 protein population is associated with snRNAs; low levels of U5 and U6 snRNAs are coimmunoprecipitated specifically with Isy1p. When the ISY1 gene was knocked out, no defect in vegetative growth was observed. Using a sensitive in vivo splicing assay, however, we observed lower splicing efficiency in the isy1 null mutant compared to wild-type, indicating that Isy1 p is important in the optimization of splicing.

Syntheses and structures of isomeric [6.6]- and [8.8]cyclophanes with 1,4-dioxabut-2-yne and 1,6-dioxahexa-2,4-diyne bridges.

All three isomers (ortho, meta, and para) of [8.8]cyclophane bearing 1,6-dioxahexa-2,4-diyne bridges have been synthesized and structually characterized by single-crystal X-ray crystallography to determine the conformation of the cyclophanes and their cavity dimensions. The three isomeric [6.6]cyclophanes bearing 1,4-dioxabut-2-yne bridges have also been synthesized from but-2-yne-1,4-diol ditosylate and the isomeric dihydroxybenzenes. The [6.6]orthocyclophane has been structurally characterized by single-crystal X-ray crystallography. The energy-minimized structures from the semiempirical AM1 calculations of these cyclophanes compare very well with the structures obtained by X-ray crystallography.

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.

Protein-RNA interactions in the U5 snRNP of Saccharomyces cerevisiae.

We present here the first insights into the organization of proteins on the RNA in the U5 snRNP of Saccharomyces cerevisiae. Photo-crosslinking with uniformly labeled U5 RNA in snRNPs reconstituted in vitro revealed five contacting proteins, Prp8p, Snu114p, p30, p16, and p10, contact by the three smaller proteins requiring an intact Sm site. Site-specific crosslinking showed that Snu114p contacts the 5' side of internal loop 1, whereas Prp8p interacts with five different regions of the 5' stem-loop, but not with the Sm site or 3' stem-loop. Both internal loops in the 5' domain are essential for Prp8p to associate with the snRNP, but the conserved loop 1 is not, although this is the region to which Prp8p crosslinks most strongly. The extensive contacts between Prp8p and the 5' stem-loop of U5 RNA support the hypothesis that, in spliceosomes, Prp8p stabilizes loop 1-exon interactions. Moreover, data showing that Prp8p contacts the exons even in the absence of loop 1 indicate that Prp8p may be the principal anchoring factor for exons in the spliceosome. This and the close proximity of the spliceosomal translocase, Snu114p, to U5 loop 1 and Prp8p support and extend the proposal that Snu114p mimics U5 loop 1 during a translocation event in the spliceosome.

Identification and functional analysis of hPRP17, the human homologue of the PRP17/CDC40 yeast gene involved in splicing and cell cycle control.

The PRP17 gene of the yeast Saccharomyces cerevisiae encodes a protein that participates in the second step of the splicing reaction. It was found recently that the yeast PRP17 gene is identical to the cell division cycle CDC40 gene. The PRP17/CDC40 gene codes for a protein with several copies of the WD repeat, a motif found in a large family of proteins that play important roles in signal transduction, cell cycle progression, splicing, transcription, and development. In this report, we describe the identification of human, nematode, and fission yeast homologues of the PRP17/CDC40 gene of S. cerevisiae. The newly identified proteins share homology with the budding yeast protein throughout their entire sequence, with the similarity being greatest in the C-terminal two thirds that includes the conserved WD repeats. We show that a yeast-human chimera, carrying the C-terminal two thirds of the hPRP17 protein, is able to complement the cell cycle and splicing defects of a yeast prp17 mutant. Moreover, the yeast and yeast-human chimeric proteins co-precipitate the intron-exon 2 lariat intermediate and the intron lariat product, providing evidence that these proteins are spliceosome-associated. These results show the functional conservation of the Prp17 proteins in evolution and suggest that the second step of splicing takes place by a similar mechanism throughout eukaryotes.

Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins.

A set of seven structurally related Sm proteins forms the core of the snRNP particles containing the spliceosomal U1, U2, U4 and U5 snRNAs. A search of the genomic sequence of Saccharomyces cerevisiae has identified a number of open reading frames that potentially encode structurally similar proteins termed Lsm (Like Sm) proteins. With the aim of analysing all possible interactions between the Lsm proteins and any protein encoded in the yeast genome, we performed exhaustive and iterative genomic two-hybrid screens, starting with the Lsm proteins as baits. Indeed, extensive interactions amongst eight Lsm proteins were found that suggest the existence of a Lsm complex or complexes. These Lsm interactions apparently involve the conserved Sm domain that also mediates interactions between the Sm proteins. The screens also reveal functionally significant interactions with splicing factors, in particular with Prp4 and Prp24, compatible with genetic studies and with the reported association of Lsm proteins with spliceosomal U6 and U4/U6 particles. In addition, interactions with proteins involved in mRNA turnover, such as Mrt1, Dcp1, Dcp2 and Xrn1, point to roles for Lsm complexes in distinct RNA metabolic processes, that are confirmed in independent functional studies. These results provide compelling evidence that two-hybrid screens yield functionally meaningful information about protein-protein interactions and can suggest functions for uncharacterized proteins, especially when they are performed on a genome-wide scale.