PubMed Health⌕ Search

Biomedical subjects

B Séraphin

Publications and source records attributed to B Séraphin.

At least 55 records · Page 3Linked to original sources
PubMed1996

An efficient PCR mutagenesis strategy without gel purification [correction of "purificiation"] step that is amenable to automation.

We describe here an improved megaprimer PCR mutagenesis strategy. The cumbersome gel purification step that is usually used can be omitted by appropriately cleaving the first and second DNA templates with restriction enzymes and enzymatically removing remaining primers from the first PCR reaction. We show that this improved procedure is reproducible and highly efficient. Furthermore this method is suitable for automation because all the steps are now carried out in reaction tubes.

DNA↗
PubMed1996

Accurate processing of a eukaryotic precursor ribosomal RNA by ribonuclease MRP in vitro.

Very few of the enzymes required for eukaryotic precursor ribosomal RNA (pre-rRNA) processing have been identified. Ribonuclease (RNase) MRP was characterized as a nuclease that cleaves mitochondrial replication primers, but it is predominantly nucleolar. Previous genetic evidence revealed that this ribonucleoprotein is required, directly or indirectly, for cleavage of the yeast pre-rRNA in vivo at site A3. Here, an in vitro processing system that accurately reproduces this cleavage is described. Biochemical purification and the use of extracts depleted of the MRP RNA demonstrate that endonucleolytic cleavage of the pre-rRNA is directly mediated by RNase MRP. This establishes a role for RNase MRP in the nucleolus.

Base Sequence↗
PubMed1995

A family of Ran binding proteins that includes nucleoporins.

Ran, a small nuclear GTP binding protein, is essential for the translocation of nuclear proteins through the nuclear pore complex. We show that several proteins, including the Saccharomyces cerevisiae Nup2p and Caenorhabditis elegans F59A2.1 nucleoporins, contain domains similar to the previously characterized murine Ran binding protein (RBP, termed RBP1). To test the significance of this similarity, we have used the corresponding domains of Nup2p and a putative S. cerevisiae RBP in Ran binding assays and the yeast two-hybrid system. Both proteins bind S. cerevisiae Ran, but only the putative S. cerevisiae RBP binds human Ran. Two-hybrid analysis revealed Ran-Ran interactions and that yeast and human Rans can interact. These data identify Nup2p as a target for Ran in the nuclear pore complex, suggesting a direct role for it in nuclear-cytoplasmic transport. We discuss the possibility that proteins harboring Ran binding domains link the Ran GTPase cycle to specific functions in the nucleus.

Amino Acid Sequence↗
PubMed1995

Sm and Sm-like proteins belong to a large family: identification of proteins of the U6 as well as the U1, U2, U4 and U5 snRNPs.

Several small nuclear RNAs (snRNAs), including the spliceosomal U1, U2, U4 and U5 snRNAs, are associated with Sm proteins. These eight small proteins form a heteromeric complex that binds to snRNAs and plays a major role in small nuclear ribonucleoprotein (snRNP) biogenesis and transport. These proteins are also a major target for autoantibodies in the human disease systemic lupus erythematosus. By sequence comparison I have shown that all the known Sm proteins share a common structural motif which might explain their immunological cross-reactivity. Database searches using this motif uncovered a large number of Sm-like proteins from plants, animals and fungi. These proteins have been grouped in at least 13 different subfamilies. Genes encoding divergent yeast members were cloned and used to produce tagged fusion proteins. Some of these proteins are canonical Sm proteins as they associate with the yeast U1, U2, U4/U6 and U5 snRNAs. Surprisingly, one Sm-like protein was found to be a component of the U6 snRNP. These findings have implications for the structure of the Sm protein complex, spliceosomal snRNP evolution, snRNA transport and modification as well as the involvement of Sm proteins in systemic lupus erythematosus.

Amino Acid Sequence↗
PubMed1994

The yeast BDF1 gene encodes a transcription factor involved in the expression of a broad class of genes including snRNAs.

While screening for genes that affect the synthesis of yeast snRNPs, we identified a thermosensitive mutant that abolishes the production of a reporter snRNA at the non-permissive temperature. This mutant defines a new gene, named BDF1. In a bdf1-1 strain, the reporter snRNA synthesized before the temperature shift remains stable at the non-permissive temperature. This demonstrates that the BDF1 gene affects the synthesis rather than the stability of the reporter snRNA and suggests that the BDF1 gene encodes a transcription factor. BDF1 is present in single copy on yeast chromosome XII, and is important for normal vegetative growth but not essential for cell viability. bdf1 null mutants share common phenotypes with several mutants affecting general transcription and are defective in snRNA production. BDF1 encodes a protein of 687 amino-acids containing two copies of the bromodomain, a motif also present in other transcription factors as well as a new conserved domain, the ET domain, also present in Drosophila and human proteins.

Amino Acid Sequence↗
PubMed1994

The POP1 gene encodes a protein component common to the RNase MRP and RNase P ribonucleoproteins.

Two forms of the yeast 5.8S rRNA are generated from a large precursor by distinct processing pathways. Cleavage at site A3 is required for synthesis of the major, short form, designated 5.8S(S), but not for synthesis of the long form, 5.8S(L). To identify components required for A3 cleavage, a bank of temperature-sensitive lethal mutants was screened for those with a reduced ratio of 5.8S(S):5.8S(L). The pop1-1 mutation (for processing of precursor RNAs) shows this phenotype and also inhibits A3 cleavage. The pre-rRNA processing defect of pop1-1 strains is similar to that reported for mutations in the RNA component of RNase MRP; we show that a mutation in the RNase MRP RNA also inhibits cleavage at site A3. This is the first site shown to require RNase MRP for cleavage in vivo. The pop1-1 mutation also leads to a block in the processing of pre-tRNA that is identical to that reported for mutations in the RNA component of RNase P. The RNA components of both RNase MRP and RNase P are underaccumulated in pop1-1 strains at the nonpermissive temperature, and immunoprecipitation demonstrates that POP1p is a component of both ribonucleoproteins. The POP1 gene encodes a protein with a predicted molecular mass of 100.5 kD and is essential for viability. POP1p is the first protein component of the nuclear RNase P or RNase MRP for which the gene has been cloned.

Amino Acid Sequence↗
PubMed1993

Involvement of U6 snRNA in 5' splice site selection.

Two models describing the interaction between U6 small nuclear RNA (snRNA) and the 5' splice site of introns have been proposed on the basis of cross-linking experiments. Here it is shown that a conserved sequence present in U6 snRNA forms base pairs with conserved nucleotides at the 5' splice junction and that this interaction is involved in 5' splice site choice. These results demonstrate a specific function for U6 snRNA in splicing and suggest that U6 snRNA has a proofreading role during splice site selection. A model is presented in which this new interaction, in concert with previously described interactions between U6 snRNA, U2 snRNA, and the pre-messenger RNA, would position the branch point near the 5' splice site for the catalysis of the first splicing step.

Base Sequence↗
PubMed1993

3' splice site recognition in S. cerevisiae does not require base pairing with U1 snRNA.

The conserved nucleotides 9 and 10 of U1 small nuclear RNA (snRNA) have been proposed to base pair with either 5' exon or 3' splice site sequences. In S. pombe, U1 snRNA pairing with the conserved 3' splice site is required for the first step of splicing and viability. In contrast, we show that S. cerevisiae U1 mutants at positions 9 and 10 are fully functional. Splicing of several genes is normal in these strains, ruling out an essential base pairing between U1 snRNA and 3' splice sites. U1 snRNA positions 9 and 10 are shown to be involved in 5' splice site selection through their interaction with exon sequences. Our results demonstrate that some snRNA-pre-mRNA interactions are not evolutionarily conserved and that 3' splice site recognition occurs by different mechanisms in various organisms.

Base Sequence↗
PubMed1993

Small nuclear RNAs in messenger RNA and ribosomal RNA processing.

Ribonucleoproteins (RNPs) play essential roles in many aspects of gene expression. Two families of nuclear RNPs are involved in the processing of primary transcripts made by RNA polymerases I and II (pol I and II), two of the three polymerases present in the nuclei of eukaryotic cells. Ribosomal RNA precursor transcription by pol I, subsequent processing of the precursor, and the initial steps of ribosome assembly all take place in the nucleolus. A group of nucleolar RNPs containing small RNAs (small nucleolar RNAs or snoRNAs) are involved in the posttranscriptional nucleolar events of ribosome biosynthesis. Six members of a related family of small nuclear RNAs (snRNAs) are required for the processing of mRNA precursors in the nucleoplasm. Five of these snRNAs (U1, U2, U4-6) participate in the removal of intervening sequences while the sixth (U7) plays an essential role in the 3' processing of a subset of mRNA precursors, the histone pre-mRNAs. This is a review of structural and functional aspects of the U1-U7 snRNAs and of snoRNAs.

Base Sequence↗
PubMed1992

The yeast mitochondrial intron aI5 alpha: associated endonuclease activity and in vivo mobility.

By analyzing crosses between yeast strains carrying different combinations of mitochondrial (mt) introns, we have shown that the aI5 alpha intron is mobile in vivo. Furthermore, we have observed that the mobility of intron aI5 alpha is affected by both the nuclear and mt genotypes. We have also detected a restriction endonuclease (ENase) activity that cleaves intronless mt genomes close to the aI5 alpha intron insertion site and thus might be involved in intron mobility. This is further supported by the fact that this ENase activity is only detected in a strain containing the aI5 alpha intron. Furthermore, similar to other ENases encoded by mobile mt introns of yeast, the ENase generates a cut with a four-base 3'-OH overhang. Thus, intron aI5 alpha represents a characteristic member of the family of mobile group-I introns.

Base Sequence↗
PubMed1992

The HIT protein family: a new family of proteins present in prokaryotes, yeast and mammals.

By comparing the sequence of a putative translation product from a Saccharomyces cerevisiae split gene with several data-bases, I have uncovered a new protein family. Members of this family are found in prokaryotes as well as in lower and higher eukaryotes. The function of these proteins is unknown but they share a characteristic histidine triad that may be involved in zinc binding. This group of protein has been named the HIT protein family.

Amino Acid Sequence↗
PubMed1991

Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly.

The pre-mRNA splicing pathway is highly conserved from yeast (S. cerevisiae) to mammals. Of the four snRNPs involved in splicing three (U1, U2 and U4/U6) have been shown to be essential for in vitro splicing. To examine the remaining snRNP, we utilized our previously described genetic procedures (Seraphin and Rosbash, 1989) to prepare yeast extracts depleted of U5 snRNP. The results show that U5 snRNP is necessary for both steps of pre- mRNA splicing and for proper spliceosome assembly, i.e., addition of the U4/U5/U6 triple snRNP. The prior steps of U1 and U2 snRNP addition occur normally in the absence of U5 snRNP.

Blotting, Northern↗
PubMed1991

The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA.

Commitment complexes contain U1 snRNP as well as pre-mRNA and are the earliest functional complexes that have been described during in vitro spliceosome assembly. We have used a gel retardation assay to analyze the role of the yeast pre-mRNA cis-acting sequences in commitment complex formation. The results suggest that only a proper 5' splice site sequence is required for efficient U1 snRNA-pre-mRNA complex formation. A role for the highly conserved UACUAAC branchpoint sequence is indicated, however, by competition experiments and by the direct analysis of branchpoint mutant substrates, which cannot form one of the two commitment complex species observed with wild-type substrates. The results suggest that the formation of a U1 snRNP-pre-mRNA complex is not dependent upon the presence of a branchpoint sequence but that the branchpoint sequence is recognized prior to U2 snRNP addition during in vitro spliceosome assembly.

Actins↗
PubMed1991

Who's on first? The U1 snRNP-5' splice site interaction and splicing.

U1 small nuclear ribonucleoprotein (snRNP) is important for pre-mRNA splicing both in yeast (Saccharomyces cerevisiae) and mammalian systems. The RNA component of U1 snRNP, U1 snRNA, interacts by base pairing with pre-mRNA 5' splice sites. This article examines recent evidence suggesting that U1 snRNP is important for an early step in spliceosome assembly rather than a late step that contributes to the specificity of 5' splice-site cleavage.

Animals↗