ABSTRACT
The bluF gene of Rhodobacter capsulatus is the first gene of the bluFEDCB operon which is involved in late steps of the cobalamin synthesis. To determine the function of the bluF gene product, a bluF::omega-Km mutant strain was constructed and characterized. This vitamin B12 auxotrophic mutant strain shows a 3.5-times higher vitamin B12 requirement under phototrophic growth conditions than under chemotrophic growth conditions. Surprisingly, the bluF promoter activity does not respond to alterations to the oxygen tension or vitamin B12 concentration.
Subject(s)
Bacterial Proteins/genetics , Cobamides/metabolism , Rhodobacter capsulatus/genetics , Vitamin B 12/biosynthesis , Bacterial Proteins/metabolism , Base Sequence , DNA, Bacterial , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Molecular Sequence Data , Promoter Regions, Genetic , Rhodobacter capsulatus/metabolismABSTRACT
Mammalian splicing factor SF3a consists of three subunits of 60, 66, and 120 kDa and functions early during pre-mRNA splicing by converting the U2 snRNP into its active form. A cDNA encoding the 120-kDa subunit of SF3a has been cloned. The SF3a120 gene was localized to human chromosome 22, and three mRNAs of 3.2, 3.8, and 5.7 kb are ubiquitously expressed. The N-terminal half of the deduced SF3a120 amino acid sequence contains a tandemly repeated motif (the SURP module) that has recently been identified in the essential splicing factor PRP21p of Saccharomyces cerevisiae, the Drosophila alternative splicing regulator suppressor-of-white-apricot, and four proteins from nematodes and mammals; the C-terminal half is organized into a proline-rich region and a ubiquitin-like domain. The spacing between the SURP modules and the protein's essential function in constitutive splicing identify SF3a120 as the mammalian homologue of yeast PRP21p. Binding studies with truncated derivatives of SF3a120 revealed that the SURP domains function in binding to SF3a60, whereas a region of 130 amino acids C-terminal to these domains is essential for contacts with SF3a66.
Subject(s)
Fungal Proteins/genetics , Multigene Family , RNA Splicing , RNA-Binding Proteins/genetics , Ribonucleoprotein, U2 Small Nuclear , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 22 , Cloning, Molecular , Cricetinae , DNA, Complementary/genetics , Female , Humans , Male , Mice , Molecular Sequence Data , Protein Binding , Protein Conformation , Repetitive Sequences, Nucleic Acid , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
We have developed an in vitro splicing complementation assay to investigate the domain structure of the mammalian U4 small nuclear RNA (snRNA) through mutational analysis. The addition of affinity-purified U4 snRNP or U4 RNA to U4-depleted nuclear extract efficiently restores splicing activity. In the U4-U6 interaction domain of U4 RNA, only stem II was found to be essential for splicing activity; the 5' loop is important for spliceosome stability. In the central domain, we have identified a U4 RNA sequence element that is important for splicing and spliceosome assembly. Surprisingly, an intact Sm domain is not essential for splicing in vitro. Our data provide evidence that several distinct regions of U4 RNA contribute to snRNP assembly, spliceosome assembly and stability, and splicing activity.
Subject(s)
Cell Nucleus/metabolism , RNA Splicing/genetics , RNA, Small Nuclear/genetics , Ribonucleoproteins/genetics , Base Sequence , Cell-Free System/metabolism , DNA Mutational Analysis , Genetic Complementation Test , HeLa Cells , Humans , Molecular Sequence Data , Nucleic Acid Conformation , Ribonucleoproteins, Small Nuclear , Structure-Activity RelationshipABSTRACT
We have analyzed the assembly of the spliceosomal U4/U6 snRNP by injecting synthetic wild-type and mutant U4 RNAs into the cytoplasm of Xenopus oocytes and determining the cytoplasmic-nuclear distribution of U4 and U4/U6 snRNPs by CsCl density gradient centrifugation. Whereas the U4 snRNP was localized in both the cytoplasmic and nuclear fractions, the U4/U6 snRNP was detected exclusively in the nuclear fraction. Cytoplasmic-nuclear migration of the U4 snRNP did not depend on the stem II nor on the 5' stem-loop region of U4 RNA. Our data provide strong evidence that, following the cytoplasmic assembly of the U4 snRNP, the interaction of the U4 snRNP with U6 RNA/RNP occurs in the nucleus; furthermore, cytoplasmic-nuclear transport of the U4 snRNP is independent of U4/U6 snRNP assembly.
Subject(s)
Cell Nucleus/metabolism , Ribonucleoproteins/genetics , Animals , Base Sequence , Biological Transport , Cytoplasm/metabolism , Humans , Microinjections , Molecular Sequence Data , Nucleic Acid Conformation , RNA/genetics , Ribonucleoproteins/metabolism , Ribonucleoproteins, Small Nuclear , Transcription, Genetic , Xenopus laevisABSTRACT
U4 snRNA is phylogenetically highly conserved and organized in several domains. To determine the function of each of the domains of human U4 snRNA in the multi-step process of snRNP and spliceosome assembly, we used reconstitution procedures in combination with snRNA mutagenesis. The highly conserved 5' terminal domain of U4 snRNA consists of the stem I and stem II regions that have been proposed to base pair with U6 snRNA, and the 5' stem-loop structure. We found that each of these structural elements is essential for spliceosome assembly. However, only the stem II region is required for U4-U6 interaction, and none of these elements for Sm protein binding. In contrast, the 3' terminal domain of U4 snRNA containing the Sm binding site is dispensable for both U4-U6 interaction and spliceosome assembly. Our results support an organization of the U4 snRNP into multiple functional domains, each of which acts at distinct stages of snRNP and spliceosome assembly.
