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P Legrain

Publications and source records attributed to P Legrain.

At least 37 records · Page 2Linked to original sources
PubMed1997

In vivo commitment to splicing in yeast involves the nucleotide upstream from the branch site conserved sequence and the Mud2 protein.

Pre-mRNA splicing is a stepwise nuclear process involving intron recognition and the assembly of the spliceosome followed by intron excision. We previously developed a pre-mRNA export assay that allows the discrimination between early steps of spliceosome formation and splicing per se. Here we present evidence that these two assays detect different biochemical defects for point mutations. Mutations at the 5' splice site lead to pre-mRNA export, whereas 3' splice site mutations do not. A genetic screen applied to mutants in the branch site region shows that all positions in the conserved TACTAAC sequence are important for intron recognition. An exhaustive analysis of pre-mRNA export and splicing defects of these mutants shows that the in vivo recognition of the branch site region does not involve the base pairing of U2 snRNA with the pre-mRNA. In addition, the nucleotide preceding the conserved TACTAAC sequence contributes to the recognition process. We show that a T residue at this position allows for optimal intron recognition and that in natural introns, this nucleotide is also used preferentially. Moreover, the Mud2 protein is involved in the recognition of this nucleotide, thus establishing a role for this factor in the in vivo splicing pathway.

Base Composition↗
PubMed1997

Toward a functional analysis of the yeast genome through exhaustive two-hybrid screens.

The genome of the yeast Saccharomyces cerevisiae is now completely sequenced. Despite successful genetic work in recent years, 60% of yeast genes have no assigned function and half of those encode putative proteins without any homology with known proteins. Genetic analyses, such as suppressor or synthetic lethal screens, have suggested many functional links between gene products, some of which have been confirmed by biochemical means. Altogether, these approaches have led to a fairly extensive knowledge of defined biochemical pathways. However, the integration of these pathways against the background of complexity in a living cell remains to be accomplished. The two-hybrid method applied to the yeast genome might allow the characterization to the network of interactions between yeast proteins, leading to a better understanding of cellular functions. Such an analysis has been performed for the bacteriophage T7 genome that encodes 55 proteins and for Drosophila cell cycle regulators. However, the currently available two-hybrid methodology is not suitable for a large-scale project without specific methodological improvements In particular, the exhaustivity and selectivity of the screens must first be greatly improved. We constructed a new yeast genomic library and developed a highly selective two-hybrid procedure adapted for exhaustive screens of the yeast genome. For each bait we selected a limited set of interacting preys that we classified in categories of distinct heuristic values. Taking into account this classification, new baits were chosen among preys and, in turn, used for second-round screens. Repeating this procedure several times led to the characterization of the network of interactions. Using known pre-mRNA splicing factors as initial baits, we were able to characterize new interactions between known splicing factors, identify new yeast splicing factors, including homologues of human SF1 and SAP49, and reveal novel potential functional links between cellular pathways. Using different cellular pathways as anchor points, this novel strategy allows us to envision the building of an interaction map of the yeast proteome. In addition, this two-hybrid strategy could be applied to other genomes and might help to resolve the human protein linkage map.

Amino Acid Sequence↗
PubMed1996

Essential domains of the PRP21 splicing factor are implicated in the binding to PRP9 and PRP11 proteins and are conserved through evolution.

The yeast Prp9p, Prp11p, Prp21p proteins form a multimolecular complex identified as the SF3a splicing factor in higher eukaryotes. This factor is required for the assembly of the prespliceosome. Prp21p interacts with both Prp9p and Prp11p, but the molecular basis of these interactions is unknown. Prp21p, its human homologue, and the so-called SWAP proteins share a tandemly repeated motif, the surp module. Given the evolutionary conservation and the role of SWAP proteins as splicing regulators, it has been proposed that surp motifs are essential for interactions between Prp21p and other splicing factors. In order to characterize functional domains of Prp21p and to identify potential additional functions of this protein, we isolated a series of heat-sensitive prp21 mutants. Our results indicate that prp21 heat-sensitive mutations are associated with defects in the interaction with Prp9p, but not with Prp11p. Interestingly, most heat-sensitive point mutants associate a strong splicing defect with a pre-mRNA nuclear export phenotype, as does the prp9-1 heat-sensitive mutant. Deletion analyses led to the definition of domains required for viability. These domains are responsible for the interaction with Prp9p and Prp11p and are conserved through evolution. They do not include the most conserved surp1 module, suggesting that the conservation of this motif in two families of proteins may reflect a still unknown function dispensable in yeast under standard conditions.

Amino Acid Sequence↗
PubMed1995

Homodimerization of the human U1 snRNP-specific protein C.

The U1 snRNP-specific protein C contains an N-terminal zinc finger-like CH motif which is required for the binding of the U1C protein to the U1 snRNP particle. Recently a similar motif was reported to be essential for in vivo homodimerization of the yeast splicing factor PRP9. In the present study we demonstrate that the human U1C protein is able to form homodimers as well. U1C homodimers are found when (i) the human U1C protein is expressed in Escherichia coli, (ii) immunoprecipitations with anti-U1C antibodies are performed on in vitro translated U1C, and when (iii) the yeast two hybrid system is used. Analyses of mutant U1C proteins in an in vitro dimerization assay and the yeast two hybrid system revealed that amino acids within the CH motif, i.e. between positions 22 and 30, are required for homodimerization.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Splicing factor SF3a60 is the mammalian homologue of PRP9 of S.cerevisiae: the conserved zinc finger-like motif is functionally exchangeable in vivo.

A cDNA encoding the 60 kDa subunit of mammalian splicing factor SF3a has been isolated. The deduced protein sequence reveals a 30% identity to the PRP9 splicing protein of the yeast S.cerevisiae. The highest homology is present in a zinc finger-like region in the C-terminal domain of both proteins. The PRP9 zinc finger-like motif has been replaced by the equivalent region of mammalian SF3a60. The chimeric protein rescues the temperature-sensitive phenotype of the prp9-1 mutant strain demonstrating that not only the structure but also the function of this domain has been conserved during evolution.

Amino Acid Sequence↗
PubMed1994

Interaction between the first and last nucleotides of pre-mRNA introns is a determinant of 3' splice site selection in S. cerevisiae.

The splicing of group II and nuclear pre-mRNAs introns occurs via a similar splicing pathway and some of the RNA-RNA interactions involved in these splicing reactions show structural similarities. Recently, genetic analyses performed in a group II intron and the yeast nuclear actin gene suggested that non Watson-Crick interactions between intron boundaries are important for the second splicing step efficiency in both classes of introns. We here show that, in the yeast nuclear rp51A intron, a G to A mutation at the first position activates cryptic 3' splice sites with the sequences UAC/ or UAA/. Moreover, the natural 3' splice site could be reactivated by a G to C substitution of the last intron nucleotide. These results demonstrate that the interaction between the first and last intron nucleotides is a conserved feature of nuclear pre-mRNA splicing in yeast and is involved in the mechanism of 3' splice site selection.

Base Sequence↗
PubMed1994

Complete DNA sequence of yeast chromosome XI.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Base Sequence↗
PubMed1993

Interaction between PRP11 and SPP91 yeast splicing factors and characterization of a PRP9-PRP11-SPP91 complex.

Several proteins are involved in the early steps of the spliceosome assembly pathway. Protein-protein interactions have been identified between two Saccharomyces cerevisiae yeast splicing factors, PRP9 and SPP91. Here it is demonstrated that protein-protein interactions occur between SPP91 and PRP11. The combination of the prp9-1 mutant and a truncated prp11 mutant exhibits a synthetic lethal phenotype, suggestive of a common biochemical defect. The PRP9 and PRP11 proteins do not interact directly, but the PRP9 and PRP11 molecules can simultaneously bind SPP91 to form a three-molecule complex. Structurally and functionally related proteins are found in mammalian cells and are associated in a single biochemical fraction. This strongly suggests that the PRP9-SPP91-PRP11 complex is a key element of the splicing machinery.

Fungal Proteins↗
PubMed1993

Evidence that the 60-kDa protein of 17S U2 small nuclear ribonucleoprotein is immunologically and functionally related to the yeast PRP9 splicing factor and is required for the efficient formation of prespliceosomes.

Small nuclear ribonucleoprotein (snRNP) U2 functions in the splicing of mRNA by recognizing the branch site of unspliced mRNA. The binding of U2 snRNP and other components to pre-mRNA leads to the formation of a stable prespliceosome. In HeLa nuclear extracts, U2 snRNP exists either as a 17S form (under low salt conditions) or a 12S form (at higher salt concentrations). We have recently shown that the purified 17S U2 snRNP contains nine proteins with apparent molecular masses of 35, 53, 60, 66, 92, 110, 120, 150, and 160 kDa in addition to the common snRNP proteins and the U2 proteins A' and B" that are found in the 12S U2 snRNP form. By using antibodies against the PRP9 protein from Saccharomyces cerevisiae (a protein required for the addition of U2 to prespliceosomes in yeast), we have shown that the 60-kDa protein specific to human U2 snRNP particles is structurally related to the yeast PRP9 protein. Interestingly, anti-PRP9 antibodies strongly inhibit prespliceosome formation in HeLa nuclear splicing extracts, resulting in a complete inhibition of the mRNA splicing reaction in vitro. This indicates that the U2 60-kDa protein may also be functionally related to its yeast counterpart PRP9. Most importantly, the addition of purified 17S U2 snRNPs, but not of 12S U2 snRNPs, to HeLa splicing extracts in which the endogeneous U2 snRNPs have been functionally neutralized with anti-PRP9 antibodies fully restores the mRNA-splicing activity of the extracts. These data suggest further that the 17S form is the functionally active form of U2 snRNP in the spliceosome.

Cell Nucleus↗
PubMed1993

The biochemical defects of prp4-1 and prp6-1 yeast splicing mutants reveal that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP.

We have raised specific antibodies against the PRP6 protein and shown that the U4, U5 and U6 snRNAs are co-precipitated with this protein. Using splicing extracts prepared from in vivo heat-inactivated cells, we have characterized the prp4-1 and prp6-1 biochemical defects. In inactivated prp4-1 cell extracts, the U6 snRNA content as well as the U6, U4/U6 snRNPs and the [U4/U6.U5] tri-snRNP particles amounts are severely reduced. In inactivated prp6-1 cell extracts, the PRP6 mutant protein is barely detectable. Glycerol gradient analyses indicate that, in these extracts, the [U4/U6.U5] tri-snRNPs are present in very low amounts, but U4/U6 snRNP particles are normally represented. These results establish that the PRP6 protein is required for the accumulation of the [U4/U6.U5] tri-snRNP. We found no evidence for the presence of the PRP6 protein in the U4/U6 particle.

Centrifugation, Density Gradient↗
PubMed1993

Interactions between PRP9 and SPP91 splicing factors identify a protein complex required in prespliceosome assembly.

The PRP9 protein is a yeast splicing factor implicated in the early steps of spliceosome assembly whose sequence contains an amino-terminal putative leucine zipper structure and two carboxy-terminal motifs reminiscent of zinc fingers. Here, we show that the deletion of the second carboxy-terminal motif results in a dominant lethal phenotype. This observation, combined with an in vivo-binding assay for protein-protein interactions, reveals the presence of two distinct binding sites on the PRP9 protein. The carboxy-terminal region contributes to the PRP9 homodimerization, whereas the amino-terminal region binds the SPP91 splicing factor. Further experiments suggest that other factors bind to PRP9 and SPP91 proteins. Finally, we demonstrate that the PRP9 protein acts after the formation of the U1 snRNP-pre-mRNA complex. The existence of a protein complex including the PRP9 factor is discussed.

Binding Sites↗
PubMed1992

A novel gene, spp91-1, suppresses the splicing defect and the pre-mRNA nuclear export in the prp9-1 mutant.

Processing and export of nuclear pre-mRNA are believed to be competing processes in the nucleus. In order to identify factors which are involved in these processes, we isolated suppressors that relieve the growth defect of a prp9-1 temperature-sensitive mutant strain of Saccharomyces cerevisiae. The prp9-1 mutation was previously shown to abolish splicing and to target pre-mRNA to the cytoplasm. One of the suppressors, spp91-1, corrects the prp9-1 growth defect through partial restoration of splicing and by a complete reversion of the pre-mRNA escape phenotype. This suppressor is specific for two prp9 alleles and cannot substitute for PRP9 function. The mutant and wild-type alleles of SPP91 were cloned and sequenced. SPP91 encodes a novel protein essential for mitotic growth whose sequence contains motifs indicative of a nuclear localization. In vivo depletion of SPP91 in a prp9-1 genetic background is lethal and is associated with reduced amounts of spliced mRNA and accumulation of pre-mRNA. This observation strongly supports the hypothesis that SPP91 encodes a PRP factor. We suggest that spp91-1 increases pre-mRNA retention in the nucleus by improving the formation of the spliceosome and thereby allowing a larger proportion of the pre-mRNA molecules to be spliced.

Alleles↗
PubMed1992

Sequence of a 10.7 kb segment of yeast chromosome XI identifies the APN1 and the BAF1 loci and reveals one tRNA gene and several new open reading frames including homologs to RAD2 and kinases.

We report here the DNA sequence of a segment of chromosome XI of Saccharomyces cerevisiae extending over 10.7 kb. The sequence was determined using a double-strand sequencing strategy adapted from the random-clone strategy. The segment contains seven non-overlapping long open reading frames, YKL500, 505, 510, 513, 516, 518 and 520 and one tRNA gene. YKL505 and YKL513 are two already sequenced genes, the BAF1/ABF1 and the APN1 genes, respectively. YKL510 exhibits a strong homology to the RAD2 protein and YKL516 is presumably a protein kinase.

Amino Acid Sequence↗
PubMed1991

Cloning of the two essential yeast genes, PRP6 and PRP9, and their rapid mapping, disruption and partial sequencing using a linker insertion strategy.

In the yeast Saccharomyces cerevisiae, some thermosensitive (ts) mutants have been shown to be impaired in pre-mRNA splicing (prp mutants). From a yeast genomic library, we have isolated plasmids that complement prp6 or prp9 ts mutations. These plasmids also complement the ts growth defect of additional independent mutants identified as new prp6 and prp9 ts alleles, indicating that the cloned DNAs encode PRP6 and PRP9 genes, respectively. Here, we describe the restriction maps of these loci which are localized on chromosome II and IV, respectively. The limits of open reading frames (ORFs) within the cloned inserts have been determined using a linker insertion strategy combined with the ts complementation assay. Double-strand DNA sequencing was also performed directly on the yeast expression vector from the inserted linkers. Gene disruption experiments demonstrate that both genes are essential for viability.

Base Sequence↗
PubMed1990

The molecular characterization of PRP6 and PRP9 yeast genes reveals a new cysteine/histidine motif common to several splicing factors.

prp6 and prp9 thermosensitive (ts) mutants are affected in pre-mRNA splicing and transport from the nucleus to the cytoplasm. PRP6 and PRP9 wild-type alleles have been sequenced. DNA sequence analysis reveals homologies in the 5' and 3' non-coding regions, suggesting a common regulation of gene expression. PRP6 and PRP9 genes encode a 899 amino acid and a 530 amino acid protein, respectively. The PRP6 protein has repeated motifs that evoke helix-loop-helix structures. Both PRP6 and PRP9 proteins have cysteine/histidine motifs loosely related to those found in zinc finger proteins. The substitution of some, but not all, of these residues by directed mutagenesis has a critical effect on the protein function. Homology searches reveal that two other proteins known to be involved in the nuclear splicing pathway--the yeast PRP11 and the human U1C proteins--contain similar sequences. The five cysteine/histidine motifs found in these four proteins display amino acid similarities in addition to the cysteine and histidine residues, indicating that they participate in biological structures or functions related to the splicing process. In addition, PRP6 and PRP9 exhibit leucine repeat motifs which may be implicated in protein interactions. The prp6 and prp9 ts mutations have been mapped and sequenced.

Alleles↗