ABSTRACT
The divergently transcribed yolk protein genes (Yp1 and Yp2) of Drosophila melanogaster are expressed only in adult females, in fat body tissue and in ovarian follicle cells. Using an in vitro transcription assay, we have identified a single 12-bp DNA element that activates transcription from the promoters of both Yp genes. In vivo, this regulatory element is tissue specific: it activates transcription of Yp1 and Yp2 reporter genes in follicle cells but has no detectable effect in fat body or other tissues. The sequence of the element consists of two recognition sites for the GATA family of transcription factors. We show that among the Drosophila genes known to encode GATA factors, only dGATAb is expressed in ovaries. The single transcript that we detect in ovaries is alternatively spliced or initiated to produce an ovary-specific isoform of the protein. Bacterially expressed dGATAb binds to the 12-bp element; a similar binding activity is also present in the Kc0 nuclear extracts used for in vitro transcription assays. These in vitro and in vivo results lead us to propose that dGATAb makes several developmentally regulated products, one of which is a follicle cell-specific protein activating transcription of Yp1 and Yp2 from a known regulatory element.
Subject(s)
DNA-Binding Proteins/biosynthesis , DNA-Binding Proteins/metabolism , Drosophila Proteins , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Egg Proteins/biosynthesis , Gene Expression Regulation , Nuclear Proteins/biosynthesis , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites , Blotting, Northern , Crosses, Genetic , DNA Transposable Elements , DNA-Binding Proteins/genetics , Egg Proteins/genetics , Female , Male , Mice , Molecular Sequence Data , Mutagenesis , Nuclear Proteins/genetics , Oligodeoxyribonucleotides , Ovarian Follicle/metabolism , Ovary/metabolism , Recombinant Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Sequence Deletion , Templates, Genetic , Transcription, Genetic , beta-Galactosidase/biosynthesisABSTRACT
Nuclear pre-mRNA splicing in Saccharomyces cerevisiae, as in higher eukaryotes, occurs in large RNA-protein complexes called spliceosomes. The small nuclear RNA components, U1, U2, U4, U5, and U6, have been extensively studied; however, very little is known about the protein components of yeast spliceosomes. Here we use antibodies against the precursor RNA processing protein PRP8, a protein component of the U5 small nuclear ribonucleoprotein particle, to detect its association with spliceosomes throughout the splicing reaction and in a post-splicing complex containing the excised intron. In addition, an indirect immunological approach has been developed that confirms the presence of precursor RNA processing protein PRP8 in isolated spliceosomes. This method has possible general application for the analysis of ribonucleoprotein particle complexes.
Subject(s)
RNA Precursors/genetics , RNA Splicing , RNA, Ribosomal/genetics , RNA, Small Nuclear/genetics , Ribonucleoproteins/genetics , Saccharomyces cerevisiae/genetics , Base Sequence , Molecular Sequence Data , Oligonucleotide Probes , RNA, Catalytic , RNA, Ribosomal/isolation & purification , Ribonucleoproteins/isolation & purification , Ribonucleoproteins, Small NuclearABSTRACT
While there are some differences in the nuclear pre-mRNA splicing machineries of Saccharomyces cerevisiae and higher eukaryotic cells, it is apparent that the fundamental mechanism of this reaction is highly conserved. S. cerevisiae is, therefore, an attractive organism for the study of splicing, since it is amenable to classical and molecular genetics as well as traditional biochemical methods. Here we present an outline of some of the advances which have resulted from this powerful combination of approaches.
Subject(s)
RNA Precursors/analysis , RNA Splicing , RNA, Nuclear/analysis , Saccharomyces cerevisiae/genetics , Introns , Mutation , RNA, Messenger/genetics , RibonucleoproteinsABSTRACT
Strains of Saccharomyces cerevisiae that bear the temperature-sensitive mutation rna8-1 are defective in nuclear pre-mRNA splicing at the restrictive temperature (36 degrees C), suggesting that the RNA8 gene encodes a component of the splicing machinery. The RNA8 gene was cloned by complementation of the temperature-sensitive growth defect of an rna8-1 mutant strain. Integrative transformation and gene disruption experiments confirmed the identity of the cloned DNA and demonstrated that the RNA8 gene encodes an essential function. The RNA8 gene was shown to be represented once per S. cerevisiae haploid genome and to encode a low-abundance transcript of approximately 7.4 kilobases. By using antisera raised against beta-galactosidase-RNA8 fusion proteins, the RNA8 gene product was identified in S. cerevisiae cell extracts as a low-abundance protein of approximately 260 kilodaltons. Immunodepletion of the RNA8 protein specifically abolished the activity of S. cerevisiae in vitro splicing extracts, confirming that RNA8 plays an essential role in splicing.
Subject(s)
Fungal Proteins/genetics , Genes, Fungal , RNA Precursors/genetics , RNA Splicing , Saccharomyces cerevisiae/genetics , Animals , Cloning, Molecular , DNA Restriction Enzymes , Female , Fungal Proteins/analysis , Genetic Complementation Test , Immune Sera , Immunoassay , Mutation , Nucleic Acid Hybridization , Plasmids , RNA, Fungal/genetics , Rabbits , Temperature , Transcription, Genetic , Transformation, GeneticABSTRACT
The RNA8 gene of Saccharomyces cerevisiae encodes an unusually large (260 kd) protein required for pre-mRNA splicing. Immunological procedures have been used to demonstrate that the RNA8 protein is in stable association with the small nuclear RNAs snR7L and snR7S, which are also known to be required for splicing and which are present in spliceosomal complexes. RNA8 is also involved in an ATP-dependent association with two other small nuclear RNAs, snR14 and snR6. It is proposed that this represents an ATP-dependent interaction between small nuclear ribonucleoprotein particles that precedes their entry into the spliceosome.
