ABSTRACT
The box C/D snoRNAs function in directing 2'-O-methylation and/or as chaperones in the processing of ribosomal RNA. We show here that Snu13p (15.5 kD in human), a component of the U4/U6.U5 tri-snRNP, is also associated with the box C/D snoRNAs. Indeed, genetic depletion of Snu13p in yeast leads to a major defect in RNA metabolism. The box C/D motif can be folded into a stem-internal loop-stem structure, almost identical to the 15.5 kD binding site in the U4 snRNA. Consistent with this, the box C/D motif binds Snu13p/ 15.5 kD in vitro. The similarities in structure and function observed between the U4 snRNP (chaperone for U6) and the box C/D snoRNPs raises the interesting possibility that these particles may have evolved from a common ancestral RNP.
Subject(s)
Evolution, Molecular , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/chemistry , Ribonucleoproteins, Small Nucleolar/chemistry , Spliceosomes/chemistry , Yeasts/metabolism , Base Sequence , Binding Sites , Fungal Proteins/genetics , Fungal Proteins/metabolism , Gene Deletion , HeLa Cells , Humans , Molecular Weight , Nucleic Acid Conformation , Precipitin Tests , Protein Binding , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , RNA, Small Nuclear/metabolism , RNA, Small Nucleolar/chemistry , RNA, Small Nucleolar/genetics , RNA, Small Nucleolar/metabolism , RNA-Binding Proteins/genetics , Recombinant Proteins , Regulatory Sequences, Nucleic Acid/genetics , Ribonucleoprotein, U4-U6 Small Nuclear/genetics , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Ribonucleoproteins, Small Nucleolar/genetics , Ribonucleoproteins, Small Nucleolar/isolation & purification , Ribonucleoproteins, Small Nucleolar/metabolism , Spliceosomes/genetics , Substrate Specificity , Yeasts/geneticsABSTRACT
We have determined the crystal structure of a spliceosomal RNP complex comprising the 15.5kD protein of the human U4/U6.U5 tri-snRNP and the 5' stem-loop of U4 snRNA. The protein interacts almost exclusively with a purine-rich (5+2) internal loop within the 5' stem-loop, giving an unusual RNA fold characterized by two tandem sheared G-A base pairs, a high degree of purine stacking, and the accommodation of a single RNA base, rotated out of the RNA chain, in a pocket of the protein. Apart from yielding the structure of an important entity in the pre-mRNA splicing apparatus, this work also implies a model for the complex of the 15.5kD protein with box C/D snoRNAs. It additionally suggests a general recognition principle in a novel family of RNA binding proteins.
Subject(s)
RNA, Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/chemistry , Ribonucleoproteins, Small Nuclear/metabolism , Spliceosomes/chemistry , Amino Acid Sequence , Base Pairing , Base Sequence , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Molecular Weight , Nucleic Acid Conformation , Protein Conformation , Purines/metabolism , RNA Precursors/chemistry , RNA Precursors/genetics , RNA Precursors/metabolism , RNA, Small Nuclear/chemistry , RNA, Small Nuclear/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , Ribonucleoproteins, Small Nucleolar/chemistry , Sequence Alignment , Spliceosomes/geneticsABSTRACT
The U5 small ribonucleoprotein particle (snRNP) contains various proteins involved in catalytic activities mediating conformational rearrangements of the spliceosome. We have isolated and characterized the evolutionarily highly conserved human U5 snRNP-specific protein U5-15kD. The crystal structure of U5-15kD determined at 1.4 A resolution revealed a thioredoxin-like fold and represents the first structure of a U5 snRNP-specific protein known so far. With respect to human thioredoxin the U5-15kD protein contains 37 additional residues causing structural changes which most likely form putative binding sites for other spliceosomal proteins or RNA. Moreover, a novel intramolecular disulfide bond replaces the canonical one found in the thioredoxin family. Even though U5-15kD appears to lack protein disulfide isomerase activity, it is strictly required for pre-mRNA splicing in vivo as we demonstrate by genetic depletion of its ortholog in Saccharomyces cerevisiae. Our data suggest that the previously reported involvement of its Schizosaccharomyces pombe ortholog Dim1p in cell cycle regulation is a consequence of its essential role in pre-mRNA splicing.
Subject(s)
Ribonucleoprotein, U5 Small Nuclear/genetics , Ribonucleoprotein, U5 Small Nuclear/metabolism , Schizosaccharomyces pombe Proteins , Amino Acid Sequence , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cloning, Molecular , Crystallography, X-Ray , Fungal Proteins/genetics , Fungal Proteins/metabolism , Humans , Molecular Sequence Data , Protein Conformation , RNA/metabolism , RNA Precursors/genetics , RNA Splicing , RNA, Messenger/genetics , Thioredoxins/chemistryABSTRACT
Activation of the spliceosome for splicing catalysis requires the dissociation of U4 snRNA from the U4/U6 snRNA duplex prior to the first step of splicing. We characterize an evolutionarily conserved 15.5 kDa protein of the HeLa [U4/U6.U5] tri-snRNP that binds directly to the 5' stem-loop of U4 snRNA. This protein shares a novel RNA recognition motif with several RNP-associated proteins, which is essential, but not sufficient for RNA binding. The 15.5kD protein binding site on the U4 snRNA consists of an internal purine-rich loop flanked by the stem of the 5' stem-loop and a stem comprising two base pairs. Addition of an RNA oligonucleotide comprising the 5' stem-loop of U4 snRNA (U4SL) to an in vitro splicing reaction blocked the first step of pre-mRNA splicing. Interestingly, spliceosomal C complex formation was inhibited while B complexes accumulated. This indicates that the 15.5kD protein, and/or additional U4 snRNP proteins associated with it, play an important role in the late stage of spliceosome assembly, prior to step I of splicing catalysis. Our finding that the 15.5kD protein also efficiently binds to the 5' stem-loop of U4atac snRNA indicates that it may be shared by the [U4atac/U6atac.U5] tri-snRNP of the minor U12-type spliceosome.
Subject(s)
RNA, Small Nuclear/metabolism , Ribonucleoproteins, Small Nuclear/genetics , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , Conserved Sequence , HeLa Cells , Humans , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Phylogeny , RNA Precursors/genetics , RNA Splicing , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Ribonucleoproteins, Small Nuclear/chemistry , Sequence Alignment , Spliceosomes/metabolismABSTRACT
We have investigated the changes in protein kinase C (PKC) activity after treatment of several cell lines with TNF. Binding studies with [3H]phorbol dibutyrate (PBt2) on whole cells revealed rapid and transient activation of PKC in Jurkat, K562, and U937 cells with a maximum of phorbol ester binding at 6 min after TNF treatment. As shown by Scatchard analysis, the TNF-induced increase of [3H]PBt2 binding reflected increments of phorbol ester binding site numbers rather than greater binding affinities. Upon subfractionation of TNF-treated U937 cells a transient increase of PBt2 binding in the membrane fraction was accompanied by a long term loss of PBt2-binding in the cytosol, indicating a TNF-induced translocation of PKC from the cytosol to the cell membrane. With histone III-S as a substrate, the determination of specific PKC activity revealed similar kinetics of PKC translocation in U937 cells. TNF also induced PKC translocation in K562 and Jurkat cells. However, although TNF caused long term down-regulation of cytosolic PKC activity in U937 cells, the cytosolic PKC activity only transiently decreased in both Jurkat and K562 cells and then recovered to near basal levels. In the human nonmalignant fibroblast cell line CCD18, PKC was not activated by TNF. Our data suggest that PKC activation may play a major role in TNF signal transduction in some, but not all target cells.
Subject(s)
Protein Kinase C/physiology , Receptors, Cell Surface/physiology , Tumor Necrosis Factor-alpha/physiology , Cell Compartmentation , Cell Membrane/enzymology , Cytosol/enzymology , Enzyme Activation , Humans , In Vitro Techniques , Phorbol 12,13-Dibutyrate/metabolism , Receptors, Tumor Necrosis Factor , Recombinant Proteins , Signal Transduction , Tumor Cells, CulturedABSTRACT
Analysis of 53 somatic cell hybrids between TNF-sensitive myeloid cells (U937) and TNF-resistant T-cell lines HUT78 (UH-hybrids) and Jurkat (UJ-hybrids), respectively, revealed complete resistance to TNF-mediated cytostasis in all cases. Moreover, all hybrids remained insensitive to a combined treatment with TNF-alpha and IFN-gamma, which exert synergistic growth inhibition and cytotoxicity on parental U937 cells. Analyses of cell surface marker expression, membrane phosphoproteins, and expression of tissue-specific cytokine genes revealed differential conservation of myeloid and T-cell-specific properties in each of these hybrids, but invariant, dominant resistance to TNF-alpha-mediated growth inhibition. All TNF-resistant hybrids expressed a membrane phosphoprotein pattern, which closely resembled that of the respective parental T-cell lines. In particular, two membrane phosphoproteins of apparent molecular weight of 50,000 and 38,000 were common in the two parental T-cell lines and all UH- and UJ-hybrid clones, suggesting a possible role of these proteins in mediating TNF resistance.
