Localisation of an Endonuclease Specific for Double-Stranded RNA within the Nucleolus and Its Implication in Processing Ribosomal Transcripts (original) (raw)
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Assays for Ribosomal RNA Processing and Ribosome Assembly
Current Protocols in Cell Biology, 2001
The synthesis of ribosomes is a major metabolic activity critical for cell growth and homeostasis. Understanding the mechanisms of ribosome biogenesis has important implications for studying both protein synthesis and cell cycle control. This unit describes several techniques for the analysis of rRNA maturation and ribosome assembly adapted for mammalian cells. Metabolic labeling of rRNA and hybridization analysis of precursors can be used to assess changes in rRNA processing that occur under experimental conditions of interest. Separation of preribosomal particles by sucrose gradient centrifugation is suitable for the analysis of proteins associated with preribosomes during their assembly and maturation in the cell nucleus.
Ribonuclease P: an enzyme with an essential RNA component
Proceedings of the National Academy of Sciences, 1978
The activity of ribonuclease P on precursor tRNA substrates from Escherichia coli can be abolished by pretreatment of this enzyme with micrococcal nuclease or pancreatic ribonuclease A, as well as by proteases and by thermal denaturation. Highly purified RNase P exhibits one prominent RNA and one prominent polypeptide com nent when examined in polyacrylamide gels containing sodum dodecyl sulfate. The buoyant density in CsCl of RNase P, 1.71 g/ml, is characteristic of a protein-RNA complex. The activity of RNase P is inhibited by various RNA molecules. The presence of a discrete RNA component in RNase P appears to be essential for enzymatic function. A model is described for enzyme-substrate recognition in which this RNA component plays an important role.
Cleavage of pre-mRNA sequences by ribonucleases bound to nuclear RNP particles of rat liver
Molecular Biology Reports, 1978
The 30S nuclear RNP particles from rat liver have been shown to split the double-stranded-(dO and single-stranded (ss) sequences of nuclear pre-mRNA. Experiments performed in vitro have demonstrated that 1) a 5'-exonuclease and an endonuclease specific for-double-stranded pre-mRNA sequences exist in the 30S pre-mRNP particles, 2) in dsRNA monophosphorylated 5'-termini arose in the course of incubation with 30S RNP and most of the products remained double-stranded. The analysis of terminal pNp nucleotides revealed a relatively high ratio of pPyp in the cleaved dsRNA, whereas the nucleosides in 5'terminal pNp of ssRNA showed nearly random distribution. Our results provide a possible explanation for the appearance of pNp termini during the processing of nuclear pre-mRNA of mammalian cells.
The Journal of Cell Biology, 2000
Nuclear RNA transcription is repressed when eukaryotic cells enter mitosis. Here, we found that the derepression of ribosomal gene (rDNA) transcription that normally takes place in telophase may be induced in prometaphase, metaphase, and anaphase mitotic HeLa cells, and therefore appears not to be dependent on completion of mitosis. We demonstrate for the first time that in vivo inhibition of the cdc2cyclin B kinase activity is sufficient to give rise to okadaic acid-sensitive dephosphorylation of the mitotically phosphorylated forms of components of the rDNA transcription machinery, and consequently to restore rDNA transcription in mitotic cells. These results, showing that during mitosis the rDNA transcription machinery is maintained repressed by the cdc2-cyclin B kinase activity, provide an in vivo demonstration of the cell cycle-dependent regulation of rDNA transcription.
An RNase P RNA subunit mutation affects ribosomal RNA processing
Nucleic Acids Research, 1996
RNase P is a ribonucleoprotein endoribonuclease responsible for the 5′ maturation of precursor tRNAs in all organisms. While analyzing mutations in conserved positions of the yeast nuclear RNase P RNA subunit, significant accumulation of an aberrant RNA of ∼193 nucleotides was observed. This abundant RNA was identified as a 3′ extended form of the 5.8S rRNA. This strain also displays a slightly elevated level of other rRNA processing intermediates with 5′-ends at processing site A2 in the internal transcribed spacer 1 (ITS1) region of the rRNA primary transcript. To test whether pre-rRNA in the region of ITS1/5.8S/ITS2 is a substrate for RNase P in vitro, nuclear RNase P was partially purified to remove contaminating nucleases. Cleavage assays were performed using an rRNA substrate transcribed in vitro which includes the 5.8S region and its surrounding processing sites in ITS1 and ITS2. Discrete cleavages of this rRNA substrate were coincident with the peak fractions of nuclear RNase P, but not with fractions corresponding to mitochondrial RNase P or ribonuclease MRP RNA. The cleavage activity is sensitive to treatment with micrococcal nuclease, also consistent with an activity attributable to RNase P. The strong RNase P cleavage sites were mapped and their possible relationships to steps in the rRNA processing pathway are considered. These observations suggest an intimate relationship between the processes of tRNA and rRNA maturation in the eukaryotic nucleus.