Biomedical subjects

N Abovich

Publications and source records attributed to N Abovich.

15 recordsLinked to original sources

PubMed2001

A biochemical function for the Sm complex.

Within the yeast commitment complex, SmB, SmD1, and SmD3 make direct contact with the pre-mRNA substrate, close to the 5' splice site. Only these three Sm proteins have long and highly charged C-terminal tails, in metazoa as well as in yeast. We replaced these proteins with tail-truncated versions. Genetic assays demonstrate that the tails contribute to similar and overlapping functions, and cross-linking assays show that the tails make direct contact with the pre-mRNA in a largely sequence-independent manner. Other biochemical assays indicate that they function at least in part to stabilize the U1 snRNP-pre-mRNA interaction. We speculate that this role may be general, and may have even evolved to aid weak intermolecular nucleic acid interactions of only a few base pairs.

Amino Acid Sequence↗

PubMed1998

A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition.

During the early events of pre-mRNA splicing, intronic cis-acting sequences are recognized and interact through a network of RNA-RNA, RNA-protein, and protein-protein contacts. Recently, we identified a branchpoint sequence binding protein in yeast (BBP). The mammalian ortholog (mBBP/SF1) also binds specifically to branchpoint sequences and interacts with the well studied mammalian splicing factor U2AF65, which binds to the adjacent polypyrimidine (PY) tract. In this paper we demonstrate that the mBBP/SF1-U2AF65 interaction promotes cooperative binding to a branchpoint sequence-polypyrimidine tract-containing RNA, and we suggest that this cooperative RNA binding contributes to initial recognition of the branchpoint sequence (BPS) during pre-mRNA splicing. We also demonstrate the essential nature of the third RBD of U2AF65 for the interaction between the two proteins, both in the presence and absence of RNA.

Binding Sites↗

PubMed1997

Identification and characterization of a yeast homolog of U1 snRNP-specific protein C.

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site.

Amino Acid Sequence↗

PubMed1997

The splicing factor BBP interacts specifically with the pre-mRNA branchpoint sequence UACUAAC.

The yeast splicing factor BBP (branchpoint bridging protein) interacts directly with pre-mRNA at or very near the highly conserved branchpoint sequence UACUAAC within the commitment complex. We also show that the recombinant protein recognizes the UACUAAC sequence. Therefore, BBP is also an acronym for branchpoint binding protein. The mammalian splicing factor SF1 is a BBP ortholog (mBBP) and an E complex component, and also has branchpoint sequence specificity. The relative conservation of this region in yeast and mammals correlates well with the RNA-binding differences between BBP and mBBP, suggesting that BBP contributes to branchpoint sequence definition in both systems.

Binding Sites↗

PubMed1997

Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals.

The commitment complex is the first defined step in the yeast (S. cerevisiae) splicing pathway. It contains U1 snRNP as well as Mud2p, which resembles human U2AF65. In a genetic screen, we identified the yeast gene MSL-5, which is a novel commitment complex component. Genetic and biochemical criteria indicate a direct interaction between Msl5p and both Mud2p and the U1 snRNP protein Prp40p. This defines a bridge between the two ends of the intron. Msl5p (renamed BBP for branchpoint bridging protein) has a mammalian ortholog, the splicing factor SF1. Our results show that SF1 interacts strongly with human U2AF65, and that SF1 is a bona fide E complex component. This implies that aspects of these novel cross-intron protein-protein interactions are conserved between yeast and mammals.

Animals↗

PubMed1996

Identification and characterization of a yeast gene encoding the U2 small nuclear ribonucleoprotein particle B" protein.

The inessential yeast gene MUD2 encodes a protein factor that contributes to U1 small nuclear ribonucleoprotein particle (snRNP)-pre-mRNA complex (commitment complex) formation. To identify other genes that contribute to this early splicing step, we performed a synthetic lethal screen with a MUD2 deletion strain. The first characterized gene from this screen, MSL1 (MUD synthetic lethal 1), encodes the yeast homolog of the well studied mammalian snRNP protein U2B". The yeast protein (YU2B") is a component of yeast U2 snRNP, and it is related to other members of the UIA-U2B" family, the human U2B" protein, the human U1A protein, and the yeast U1A protein. It binds in vitro to its RNA target, U2 snRNA stem-loop IV, without a protein cofactor, and the target resembles more closely the U1 snRNA binding site of the human U1A protein than it does the U2 snRNA binding site of human U2B". Surprisingly, the YU2B" protein lacks a C-terminal RNA binding domain, which is conserved in all other family members. Possible functional and evolutionary relationships among these proteins are discussed.

Amino Acid Sequence↗

PubMed1994

The yeast MUD2 protein: an interaction with PRP11 defines a bridge between commitment complexes and U2 snRNP addition.

In characterizing a series of yeast (Saccharomyces cerevisiae) mutants synthetic lethal with U1 RNA, we have identified a yeast gene (MUD2) with sequence similarity to the well-studied metazoan splicing factor U2AF65. The biochemical characterization indicates that the MUD2 gene product (MUD2P) contacts pre-mRNA directly and is a component of the pre-mRNA-U1 snRNP complex (commitment complex) that forms during early spliceosome assembly in yeast extracts. Unlike U1 snRNP itself, the association of MUD2P with pre-mRNA is dependent on a proper yeast branchpoint sequence. Genetic experiments indicate that MUD2P affects U2 snRNP addition. Moreover, experiments in the two-hybrid system show that PRP11P, a recently identified component of U2 snRNP, can interact directly with MUD2P. The experiments identify a specific inter-snRNP protein-protein contact that occurs during spliceosome assembly and more generally support substantial functional similarity between U2AF65 and MUD2P.

Amino Acid Sequence↗

PubMed1992

A yeast splicing factor is localized in discrete subnuclear domains.

Digital imaging microscopy has been used to visualize the splicing protein PRP6p and three other yeast nuclear proteins. The results show that PRP6p is uniquely localized to discrete subnuclear regions. A combination of cytological and biochemical assays suggests that these sites can be saturated when the protein is overexpressed and likely correspond to the location of U4/U6 snRNPs. The observations indicate that some splicing components are located in discrete subregions of the yeast nucleus, similar to the situation described for the mammalian nucleus.

Blotting, Western↗

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↗

PubMed1990

The yeast PRP6 gene encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein, and the PRP9 gene encodes a protein required for U2 snRNP binding.

PRP6 and PRP9 are two yeast genes involved in pre-mRNA splicing. Incubation at 37 degrees C of strains that carry temperature-sensitive mutations at these loci inhibits splicing, and in vivo experiments suggested that they might be involved in commitment complex formation (P. Legrain and M. Rosbash, Cell 57:573-583, 1989). To examine the specific role that the PRP6 and PRP9 products may play in splicing or pre-mRNA transport to the cytoplasm, we have characterized in vitro splicing and spliceosome assembly in extracts derived from prp6 and prp9 mutant strains. We have also characterized RNAs that are specifically immunoprecipitated with the PRP6 and PRP9 proteins. Both approaches indicate that PRP6 encodes a U4/U6 small nuclear ribonucleoprotein particle (snRNP) protein and that the PRP9 protein is required for a stable U2 snRNP-substrate interaction. The results are discussed with reference to the previously observed in vivo phenotypes of these mutants.

Genes, Fungal↗

PubMed1986

The period clock locus of D. melanogaster codes for a proteoglycan.

The period (per) gene of D. melanogaster is involved in the generation of biological rhythms. The most striking feature of the predicted coding sequence, corresponding to the key 4.5 kb transcript from this locus, is an extensive run of alternating Gly-Thr residues. This is homologous to a series of Gly-Ser repeats in a chondroitin sulfate proteoglycan. To determine whether the per transcript codes for a proteoglycan, a region of its coding sequence was expressed (in bacteria) as part of a fusion protein, which was used to immunize rabbits. When the resultant immune sera were used to probe fly protein preparations, they detected an antigen that is present in wild-type flies and absent in a per- mutant. Biochemical characterization of this antigen indicated that it is indeed a proteoglycan.

Amino Acid Sequence↗

PubMed1985

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

Diploidy↗

PubMed1985

Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-beta-galactosidase fusion.

To study the regulation of ribosomal protein genes, we constructed a 'lacZ fusion of the Saccharomyces cerevisiae RP51A gene, containing the first 64 codons of RP51A. In a strain lacking an intact RP51A gene (cells are viable due to the presence of an active RP51B gene), beta-galactosidase activity is 10-fold greater than in a strain containing RP51A. RP51A-lacZ mRNA levels are equal in the two strains, indicating that regulation is posttranscriptional. In the absence of the RP51A gene, the fusion protein is predominantly cytoplasmic and associated with polysomes, whereas in the presence of RP51A, the fusion protein is predominantly nuclear, and none is associated with polysomes. Deletions were made in the RP51A-coding portion of the fusion gene. The most extensively deleted gene, containing only the first seven RP51A codons fused to lacZ, produced a high level of beta-galactosidase activity in both the presence and the absence of the RP51A gene. In both cases, little or none of this shorter fusion protein was found associated with polysomes. Thus, a regulatory site (or sites) lies in the protein-coding region of RP51A. We suggest that posttranscriptional regulation of the rp51 fusion protein is related to assembly of the protein into ribosomes.

Escherichia coli↗

PubMed1984

A comparison of yeast ribosomal protein gene DNA sequences.

The DNA sequences of eight yeast ribosomal protein genes have been compared for the purpose of identifying homologous regions which may be involved in the coordinate regulation of ribosomal protein synthesis. A 12 bp homology was identified in the 5' DNA sequence preceding the structural gene for 6 out of 8 yeast ribosomal protein genes. In each case the homologous sequence was found at a position approximately 300 bp preceding the transcription start of the ribosomal protein gene. This homology was not identified in any non-ribosomal protein gene examined. Additional homologies between ribosomal protein genes were identified in the transcribed regions, including the untranslated 5' and 3' DNA regions flanking the coding regions.

Amino Acid Sequence↗

PubMed1984

Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes.

We cloned and sequenced the second gene coding for yeast ribosomal protein 51 (RP51B). When the DNA sequence of this gene was compared with the DNA sequence of RP51A (J.L. Teem and M. Rosbash, Proc. Natl. Acad. Sci. U.S.A. 80:4403--4407, 1983), the following conclusions emerged: both genes code for a protein of 135 amino acids; both open reading frames are interrupted by a single intron which occurs directly after the initiating methionine; the open reading frames are 96% homologous and code for the same protein with the exception of the carboxy-terminal amino acid; DNA sequence homology outside of the coding region is extremely limited. The cloned genes, in combination with the one-step gene disruption techniques of Rothstein (R. J. Rothstein, Methods Enzymol. 101:202-211, 1983), were used to generate haploid strains containing mutations in the RP51A or RP51B genes or in both. Strains missing a normal RP51A gene grew poorly (180-min generation time versus 130 min for the wild type), whereas strains carrying a mutant RP51B were relatively normal. Strains carrying mutations in the two genes grew extremely poorly (6 to 9 h), which led us to conclude that RP51A and RP51B were both expressed. The results of Northern blot and primer extension experiments indicate that strains with a wild-type copy of the RP51B gene and a mutant (or deleted) RP51A gene grow slowly because of an insufficient amount of RP51 mRNA. The growth defect was completely rescued with additional copies of RP51B. The data suggest that RP51A contributes more RP51 mRNA (and more RP51 protein) than does RP51B and that intergenic dosage compensation, sufficient to rescue the growth defect of strains missing a wild-type RP51A gene, does not take place.

Base Sequence↗