Showing papers by "Murray P. Deutscher published in 2003"

Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R

Abstract: Despite their overall accuracy, errors in macromolecular processes, such as rRNA synthesis and ribosome assembly, inevitably occur. However, whether these errors are remediated and how this might be accomplished is not known. In previous work, we showed that a double mutant strain lacking both polynucleotide phosphorylase (PNPase) and RNase R activities is inviable. In the course of examining the molecular basis for this phenotype, we found that shifting a temperature-sensitive mutant strain to 42°C led to cessation of growth and loss of cell viability. Northern analysis of RNA isolated from such cells after the temperature shift revealed that fragments of 16S and 23S rRNA accumulated to a high level, and that the amount of ribosomes and ribosomal subunits decreased due to defects in ribosome assembly. rRNA fragments were not detected at 31°C or when single mutant strains were grown at 42°C. Pulse–chase analysis showed that the rRNA fragments appeared within 5 min at 42°C, and that they accumulated before the loss of cell viability. The data are consistent with a model in which PNPase and RNase R mediate a previously unknown quality control process that normally removes defective rRNAs as soon as they are generated. In the absence of these RNases, rRNA fragments accumulate, leading to interference with ribosome maturation and ultimately to cell death.

Organization of mammalian cytoplasm.

Abstract: Although the role of macromolecular interactions in cell function has attracted considerable attention, important questions about the organization of cells remain. To help clarify this situation, we used a simple protocol that measures macromolecule release after gentle permeabilization for the examination of the status of endogenous macromolecules. Treatment of Chinese hamster ovary cells with saponin under carefully controlled conditions allowed entry of molecules of at least 800 kDa; however, there were minimal effects on internal cellular architecture and protein synthesis remained at levels comparable to those seen with intact cells. Most importantly, total cellular protein and RNA were released from these cells extremely slowly. The release of actin-binding proteins and a variety of individual cytoplasmic proteins mirrored that of total protein, while marker proteins from subcellular compartments were not released. In contrast, glycolytic enzymes leaked rapidly, indicating that cells contain at least two distinct populations of cytoplasmic proteins. Addition of microfilament-disrupting agents led to rapid and extensive release of cytoplasmic macromolecules and a dramatic reduction in protein synthesis. These observations support the conclusion that mammalian cells behave as highly organized, macromolecular assemblies (dependent on the actin cytoskeleton) in which endogenous macromolecules normally are not free to diffuse over large distances.

Nuclear protein synthesis: A re-evaluation

Abstract: It has been reported that nuclei from HeLa cells are responsible for approximately 10%-15% of total cellular protein synthesis. We show here that isolated Chinese hamster ovary (CHO) and HeLa cell nuclei are essentially inactive for translation, and that the earlier results were most likely due to cytoplasmic contamination. Moreover, we suggest that the nascent polypeptides observed in nuclei of permeabilized cells may have been due to "overpermeabilization" and consequent damage to the cells. Based on this information, we conclude that nuclear protein synthesis, if it exists, is limited to less than 1% of that in cells.