Composition and functional characterization of yeast 66S ribosome assembly intermediates

The pathway and complete collection of factors that orchestrate ribosome assembly are not clear. To address these problems, we affinity purified yeast preribosomal particles containing the nucleolar protein Nop7p and developed means to separate their components. Nop7p is associated primarily with 66S preribosomes containing either 27SB or 25.5S plus 7S pre-rRNAs. Copurifying proteins identified by mass spectrometry include ribosomal proteins, nonribosomal proteins previously implicated in 60S ribosome biogenesis, and proteins not known to be involved in ribosome production. Analysis of strains mutant for eight of these proteins not previously implicated in ribosome biogenesis showed that they do participate in this pathway. These results demonstrate that proteomic approaches in concert with genetic tools provide powerful means to purify and characterize ribosome assembly intermediates.     

Functional spliceosomal A complexes can be assembled in vitro in the absence of a penta-snRNP

Two different models currently exist for the assembly pathway of the spliceosome, namely, the traditional model, in which spliceosomal snRNPs associate in a stepwise, ordered manner with the pre-mRNA, and the holospliceosome model, in which all spliceosomal snRNPs preassemble into a penta-snRNP complex. Here we have tested whether the spliceosomal A complex, which contains solely U1 and U2 snRNPs bound to pre-mRNA, is a functional, bona fide assembly intermediate. Significantly, A complexes affinity-purified from nuclear extract depleted of U4/U6 snRNPs (and thus unable to form a penta-snRNP) supported pre-mRNA splicing in nuclear extract depleted of U2 snRNPs, whereas naked pre-mRNA did not. Mixing experiments with purified A complexes and naked pre-mRNA additionally confirmed that under these conditions, A complexes do not form de novo. Thus, our studies demonstrate that holospliceosome formation is not a prerequisite for generating catalytically active spliceosomes and that, at least in vitro, the U1 and U2 snRNPs can functionally associate with the pre-mRNA, prior to and independent of the tri-snRNP. The ability to isolate functional spliceosomal A complexes paves the way to study in detail subsequent spliceosome assembly steps using purified components.     

A systematic characterization of Cwc21, the yeast ortholog of the human spliceosomal protein SRm300

Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3′-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.     

Molecular evolution of eukaryotic genomes: hemiascomycetous yeast spliceosomal introns

As part of the exploratory sequencing program Génolevures, visual scrutinisation and bioinformatic tools were used to detect spliceosomal introns in seven hemiascomycetous yeast species. A total of 153 putative novel introns were identified. Introns are rare in yeast nuclear genes (<5% have an intron), mainly located at the 5′ end of ORFs, and not highly conserved in sequence. They all share a clear non-random vocabulary: conserved splice sites and conserved nucleotide contexts around splice sites. Homologues of metazoan snRNAs and putative homologues of SR splicing factors were identified, confirming that the spliceosomal machinery is highly conserved in eukaryotes. Several introns’ features were tested as possible markers for phylogenetic analysis. We found that intron sizes vary widely within each genome, and according to the phylogenetic position of the yeast species. The evolutionary origin of spliceosomal introns was examined by analysing the degree of conservation of intron positions in homologous yeast genes. Most introns appeared to exist in the last common ancestor of present day yeast species, and then to have been differentially lost during speciation. However, in some cases, it is difficult to exclude a possible sliding event affecting a pre-existing intron or a gain of a novel intron. Taken together, our results indicate that the origin of spliceosomal introns is complex within a given genome, and that present day introns may have resulted from a dynamic flux between intron conservation, intron loss and intron gain during the evolution of hemiascomycetous yeasts.     

Protein localisation by electron microscopy reveals the architecture of the yeast spliceosomal B complex

The spliceosome assembles on a pre‐mRNA intron by binding of five snRNPs and numerous proteins, leading to the formation of the pre‐catalytic B complex. While the general morphology of the B complex is known, the spatial arrangement of proteins and snRNP subunits within it remain to be elucidated. To shed light on the architecture of the yeast B complex, we immuno‐labelled selected proteins and located them by negative‐stain electron microscopy. The B complex exhibited a triangular shape with main body, head and neck domains. We located the U5 snRNP components Brr2 at the top and Prp8 and Snu114 in the centre of the main body. We found several U2 SF3a (Prp9 and Prp11) and SF3b (Hsh155 and Cus1) proteins in the head domain and two U4/U6 snRNP proteins (Prp3 and Lsm4) in the neck domain that connects the main body with the head. Thus, we could assign distinct domains of the B complex to the respective snRNPs and provide the first detailed picture of the subunit architecture and protein arrangements of the B complex.     

Isolation and characterization of a sucrose carrier cDNA from spinach by functional expression in yeast.

Active loading of the phloem with sucrose in leaves is an essential part of the process of supplying non-photosynthetic tissues with carbon and energy. The transport is protein mediated and coupled to proton-symport, but so far no sucrose carrier gene has been identified. Using an engineered Saccharomyces cerevisiae strain, a cDNA from spinach encoding a sucrose carrier was identified by functional expression. Yeast strains that allow the phenotypic recognition of a sucrose carrier activity were constructed by expressing a cytoplasmic invertase from yeast, or the potato sucrose synthase gene, in a strain unable to transport or grow on sucrose due to a deletion in the SUC2 gene. A spinach cDNA expression library established from the poly(A)+ RNA from source leaves of spinach and cloned in a yeast expression vector yielded transformed yeast clones which were able to grow on media containing sucrose as the sole carbon source. This ability was strictly linked to the presence of the spinach cDNA clone pS21. Analysis of the sucrose uptake process in yeast strains transformed with this plasmid show a pH-dependent uptake of sucrose with a Km of 1.5 mM, which can be inhibited by maltose, alpha-phenylglucoside, carbonyl cyanide m-chlorophenylhydrazone and p-chloromercuribenzenesulfonic acid. These data are in accordance with measurements using both leaf discs and plasma membrane vesicles from leaves of higher plants. DNA sequence analysis of the pS21 clone reveals the presence of an open reading frame encoding a protein with a molecular mass of 55 kDa. The predicted protein contains several hydrophobic regions which could be assigned to 12 membrane-spanning regions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural comparison of yeast snoRNP and spliceosomal protein Snu13p with its homologs

Snu13p is a bifunctional yeast protein involved in both messenger RNA splicing as well as ribosomal RNA maturation. Snu13p initiates assembly of ribonucleoprotein particles by interacting with a conserved RNA motif called kink turn. Unlike its archaeal homolog, L7Ae, Snu13p displays differential specificity for functionally distinct kink turns. Thus, the structures of Snu13p at different functional states, including those alone and bound with RNAs, are required to understand how the protein differentially interacts with kink turns. Although the structure of the human homolog of Snu13p bound with a spliceosomal RNA is known, there has not been a report of a structure of free Snu13p. This has hindered our ability to understand the structural basis for Snu13p's substrate specificity. We report a crystal structure of free Snu13p at 1.9A and a detailed structural comparison with its homologs. We show that free Snu13p has nearly an identical conformation as that of its human homolog bound with RNA. Interestingly, both eukaryotic proteins exhibit notable structural differences in their central beta-sheets as compared to their archaeal homolog, L7Ae. The observed structural differences offer a possible explanation to the observed difference in RNA specificity between Snu13p and L7Ae.     

In vitro reconstitution and characterization of the yeast mitochondrial degradosome complex unravels tight functional interdependence

The mitochondrial degradosome (mtEXO), the main RNA-degrading complex of yeast mitochondria, is composed of two subunits: an exoribonuclease encoded by the DSS1 gene and an RNA helicase encoded by the SUV3 gene. We expressed both subunits of the yeast mitochondrial degradosome in Escherichia coli, reconstituted the complex in vitro and analyzed the RNase, ATPase and helicase activities of the two subunits separately and in complex. The results reveal a very strong functional interdependence. For every enzymatic activity, we observed significant changes when the relevant protein was present in the complex, compared to the activity measured for the protein alone. The ATPase activity of Suv3p is stimulated by RNA and its background activity in the absence of RNA is reduced greatly when the protein is in the complex with Dss1p. The Suv3 protein alone does not display RNA-unwinding activity and the 3' to 5' directional helicase activity requiring a free 3' single-stranded substrate becomes apparent only when Suv3p is in complex with Dss1p. The Dss1 protein alone does have some basal exoribonuclease activity, which is not ATP-dependent, but in the presence of Suv3p the activity of the entire complex is enhanced greatly and is entirely ATP-dependent, with no residual activity observed in the absence of ATP. Such absolute ATP-dependence is unique among known exoribonuclease complexes. On the basis of these results, we propose a model in which the Suv3p RNA helicase acts as a molecular motor feeding the substrate to the catalytic centre of the RNase subunit.     

Molecular characterization of the spliceosomal proteins U1A and U2B" from higher plants.

In addition to their role in pre-mRNA splicing, the human spliceosomal proteins U1A and U2B" are important models of how RNP motif-containing proteins execute sequence-specific RNA binding. Genes encoding U1A and U2B" have been isolated from potato and thereby provide the only evolutionary comparison available for both proteins and represent the only full-length genes encoding plant spliceosomal proteins to have been cloned and characterized. In vitro RNA binding experiments revealed the ability of potato U2B" to interact with human U2A' to enhance sequence-specific binding and to distinguish cognate RNAs of either plant or animal origin. A comparison of the sequence of U1A and U2B" proteins indicated that multiple residues which could affect RNP motif conformation probably govern the specific distinction in RNA binding by these proteins. Since human U1A modulates polyadenylation in vertebrates, the possibility that plant U1A might be exploited in the characterization of this process in plants was examined. However, unlike vertebrate U1A, neither U1A from potato nor Arabidopsis bound their own mRNA and no evidence for binding to upstream efficiency elements in polyadenylation signals was obtained, suggesting that plant U1A is not involved in polyadenylation.     

Isolation and characterization of yeast protease B.

Protease B was purified from baker's yeast. The final preparation appeared homogeneous by ultracentrifugation and electrophoresis. The S₂₀, ω value of the enzyme was 3.1 S and its molecular weight was calculated to be 31,000 from the results of sedimentation equilibrium analysis. The amino acid composition of the enzyme was also investigated. The enzyme inactivates phosphogluconate dehydrogenase and uricase, but not malate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase or hexokinase.     

Structural and functional characterization of recombinant medaka fish alpha-amylase expressed in yeast Pichia pastoris

The medaka fish α-amylase was expressed and purified. The expression systems were constructed using methylotrophic yeast Pichia pastoris, and the recombinant proteins were secreted into the culture medium. Purified recombinant α-amylase exhibited starch hydrolysis activity. The optimal pH, denaturation temperature, and K(M) and V(max) values were determined; chloride ions were essential for enzyme activity. The purified protein was also crystallized and examined by X-ray crystallography. The structure has the (α/β)(8) barrel fold, as do other known α-amylases, and the overall structure is very similar to the structure of vertebrate (human and pig) α-amylases. A novel expression plasmid was developed. Using this plasmid, high-throughput construction of an expression system by homologous recombination in P. pastoris cells, previously reported for membrane proteins, was successfully applied to the secretory protein.     

Isolation and characterization of the gene encoding yeast debranching enzyme.

Using a genetic screen aimed at identifying cellular factors involved in Ty1 transposition, we have identified a mutation in a host gene that reduces Ty1 transposition frequency. The mutant, dbr1, is also defective in the process of intron turnover. In dbr1 cells, excised introns derived from a variety of pre-mRNAs are remarkably stable and accumulate to levels exceeding that of the corresponding mRNA. The stable excised introns accumulate in the form of a lariat that is missing the linear sequences 3' of the branchpoint. The DBR1 gene has been isolated by complementation of the transposition phenotype. DBR1 is shown to encode debranching enzyme, an RNA processing activity that hydrolyzes the 2'-5' phosphodiester linkage at the branchpoint of excised intron lariats. In Saccharomyces cerevisiae, debranching enzyme plays a requisite role in the rapid turnover of excised introns, yet its function is not essential for viability.     

The use of the two-hybrid cloning in yeast for functional characterization of protein kinase MAK-V

Identification of interaction partners opens a way to direct functional characterization of proteins. Several cDNAs coding for potential partners of protein kinase MAK-V/Hunk were isolated using two-hybrid cloning in yeast. Based on the partner properties, MAK-V/Hunk was assumed to play a role in tumorigenesis and tumor progression. With the previous results of two-hybrid cloning, MAK-V/Hunk was shown to participate in vesicular transport.     

In vivo functional characterization of a yeast nucleotide sequence: construction of a mini-Mu derivative adapted to yeast

We have constructed a derivative of the bacteriophage Mu (called MudIIZZ1), which contains the lacZ gene coding for beta-galactosidase (beta Gal) and markers suited for yeast transformation (2 mu circle replication origin and LEU2). This new transposon is an efficient tool for studying the expression of cloned yeast nucleotide sequences through beta Gal-protein fusions. It is also adapted for one-step disruption experiments so that a functional map of the same sequence can be drawn. We have used this MudIIZZ1 transposon to study a 5-kb DNA fragment which had been cloned by complementation of a cold-sensitive respiration-deficient phenotype. By testing the expression of the beta Gal fusions and the disruption phenotype, we have confirmed the presence of a gene required for mitochondrial functions, and revealed another two open reading frames in the same fragment; one of these also interferes with mitochondrial biogenesis. The method is fast and reliable, and has potential for more general purposes which are discussed.     

The spliceosomal snRNAs of Caenorhabditis elegans.

Nematodes are the only group of organisms in which both cis- and trans-splicing of nuclear mRNAs are known to occur. Most Caenorhabditis elegans introns are exceptionally short, often only 50 bases long. The consensus donor and acceptor splice site sequences found in other animals are used for both cis- and trans-splicing. In order to identify the machinery required for these splicing events, we have characterized the C. elegans snRNAs. They are similar in sequence and structure to those characterized in other organisms, and several sequence variations discovered in the nematode snRNAs provide support for previously proposed structure models. The C. elegans snRNAs are encoded by gene families. We report here the sequences of many of these genes. We find a highly conserved sequence, the proximal sequence element (PSE), about 65 bp upstream of all 21 snRNA genes thus far sequenced, including the SL RNA genes, which specify the snRNAs that provide the 5' exons in trans-splicing. The sequence of the C. elegans PSE is distinct from PSE's from other organisms.     

Stoichiometry of the Sm proteins in yeast spliceosomal snRNPs supports the heptamer ring model of the core domain

Seven Sm proteins (B/B', D1, D2, D3, E, F and G proteins) containing a common sequence motif form a globular core domain within the U1, U2, U5 and U4/U6 spliceosomal snRNPs. Based on the crystal structure of two Sm protein dimers we have previously proposed a model of the snRNP core domain consisting of a ring of seven Sm proteins. This model postulates that there is only a single copy of each Sm protein in the core domain. In order to test this model we have determined the stoichiometry of the Sm proteins in yeast spliceosomal snRNPs. We have constructed seven different yeast strains each of which produces one of the Sm proteins tagged with a calmodulin-binding peptide (CBP). Further, each of these strains was transformed with one of seven different plasmids coding for one of the seven Sm proteins tagged with protein A. When one Sm protein is expressed as a CBP-tagged protein from the chromosome and a second protein was produced with a protein A-tag from the plasmid, the protein A-tag was detected strongly in the fraction bound to calmodulin beads, demonstrating that two different tagged Sm proteins can be assembled into functional snRNPs. In contrast when the CBP and protein A-tagged forms of the same Sm protein were co-expressed, no protein A-tag was detectable in the fraction bound to calmodulin. These results indicate that there is only a single copy of each Sm protein in the spliceosomal snRNP core domain and therefore strongly support the heptamer ring model of the spliceosomal snRNP core domain.     

Proteinase yscD mutants of yeast. Isolation and characterization

Mutants of the yeast Saccharomyces cerevisiae, devoid of proteinase yscD activity, were isolated by screening for the inability of mutagenized cells to hydrolyze Ac-Ala-Ala-Pro-Ala-beta-naphthylamide in situ. One of the selected mutants bears a thermolabile activity pointing to the gene called PRD1 as being the structural gene for proteinase yscD. All mutants isolated fell into one complementation group. The defect segregates 2:2 in meiotic tetrads indicating a single gene mutation, which was shown to be recessive. Diploids heterozygous for PRD1 display gene dosage. The absence of proteinase yscD did not affect mitotic growth under rich or poor growth conditions, neither mating nor ascopore formation. Also growth of mutant cells after a nutritional shift-down was not altered. Inactivation of enzymes tested which are subject to carbon-catabolite inactivation, a process proposed to be of proteolytic nature, is not affected by the absence of proteinase yscD. Protein degradation rates in growing cells, in cells under conditions of differentiation or heat shock, showed no obvious alteration in the absence of proteinase yscD activity. Also inactivation of alpha-factor pheromone was not affected by proteinase yscD absence. Normal growth of mutant cells on glycerol indicates that the enzyme is not involved in any vital event in mitochondrial biogenesis.     

Detection and characterization of chimeric yeast artificial-chromosome clones.

Methods for the construction of yeast artificial-chromosome (YAC) clones have been designed to isolate single, large (100-1000 kb) segments of chromosomal DNA. It is apparent from early experience with this cloning system that the major artifact in YAC clones involves the formation of YACs that contain two or more unrelated pieces of DNA. Such "chimeric" YACs are not easily recognized, particularly in libraries constructed from the total DNA of an organism. In some libraries, they have been found to constitute a major fraction of the clones. Here we discuss some of our experiences with chimeric YACs, with particular emphasis on the approaches that we have employed to detect such aberrant clones. In addition, we describe the detailed characterization of one chimeric YAC isolated from a library prepared from total human DNA. The organization of this clone indicates that it formed by in vivo recombination, presumably in yeast, between two Alu sequences located on unrelated segments of human DNA.