ABSTRACT
Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Delta cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Delta cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.
Subject(s)
Protein Kinases/metabolism , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Helicases/physiology , RNA, Messenger/metabolism , RNA, Ribosomal/metabolism , Blotting, Northern , Canavanine/pharmacology , Cell Nucleus/metabolism , Conserved Sequence , Cycloheximide/pharmacology , DEAD-box RNA Helicases , Dose-Response Relationship, Drug , Gene Deletion , Models, Biological , Mutation , Oligonucleotides/metabolism , Paromomycin/pharmacology , Phenotype , Plasmids/metabolism , Point Mutation , Protein Binding , Protein Biosynthesis , Protein Kinases/genetics , Protein Kinases/physiology , Protein Structure, Tertiary , Protein Synthesis Inhibitors/pharmacology , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins , Subcellular Fractions , Time Factors , Trans-Activators , Two-Hybrid System TechniquesABSTRACT
Localization of PTH/PTH-related peptide (PTH/PTHrP) receptor mRNA and PTH induction of c-fos expression were examined in bones of 4-week-old rats by in situ hybridization. Receptor transcripts were most highly expressed by growth plate chondrocytes from lower proliferating to upper hypertrophic cell layers. PTH/PTHrP receptor mRNA also was expressed in osteoblasts as well as in some bone marrow stromal cells. Subcutaneous administration of human PTH-(1-84) (225 micrograms/kg) induced rapid, transient, and sequential expression of the protooncogene c-fos mRNA in cells in bone. Osteoblasts and chondrocytes expressing PTH/PTHrP receptor transcripts as well as some stromal cells expressed c-fos mRNA first (15-60 min), followed by transient expression in the majority of stromal cells and in osteoclasts (1-2 h). Delayed and transient induction of c-fos in cells with few or no detectable receptor transcripts suggests that PTH acts indirectly on stromal cells and osteoclasts by either stimulating osteoblasts to secrete a substance(s) that acts locally and/or inducing changes in cell-cell contacts between osteoblasts and adjacent cells.
Subject(s)
Bone and Bones/metabolism , Parathyroid Hormone/pharmacology , Proto-Oncogene Proteins c-fos/metabolism , RNA, Messenger/metabolism , Receptors, Parathyroid Hormone/metabolism , Animals , Animals, Newborn , Bone and Bones/cytology , Cells, Cultured , Growth Plate/cytology , Growth Plate/metabolism , In Situ Hybridization , Male , Rats , Tissue DistributionABSTRACT
We characterized cells that express parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor mRNA in bones of fetal and postnatal rats by in situ hybridization. During endochondral development of fetal bones, PTH/PTHrP receptor transcripts were highly expressed both in maturing chondrocytes and in osteoblasts in the periosteum and ossification center, but not in fully hypertrophic chondrocytes. Similar to the localization in the fetal bones, PTH/PTHrP receptor mRNA expression was highly localized to maturing chondrocytes in the articular cartilage and growth plate, and to osteoblasts in the femur of young rats. In both young and fetal rats, transcripts for Type X collagen were localized to hypertrophic chondrocytes, mostly between chondrocytes and bone cells both of which express PTH/PTHrP receptor mRNA. Transcripts for PTH/PTHrP receptors and alkaline phosphatase co-localized in the bone of young rats, but they did not co-localize in fetal bones at the early stages of endochondral ossification. These results show that PTH/PTHrP receptor mRNA is expressed in a cell-type and stage-specific manner during skeletal development.
