Stability of nuclear RNA in mammalian cells

Brandhorst, Bruce P.; McConkey, Edwin H.

doi:10.1016/0022-2836(74)90444-6

Cited by 193 publications

(82 citation statements)

References 32 publications

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“…9~ lo4 nucleotides/s for rRNA. Assuming a processing efficiency of 2 % for mRNA [71] and 51 % for rRNA [70], the overall rate of transcription required is 0.3 -1.1 x lo6 nucleotides/s for mRNA and 0.05-0.11 x lo6 nucleotides/s for rRNA. Given a constant rate of RNA chain elongation of 83 nucleotides/s [72,73] for 2 x lo4 molecules of form A, we can calculate a total polymerization rate of 1.66 x lo6 nucleotides/s which is more than 10 times the calculated maximum required to maintain cytoplasmic levels of rRNA.…”

Section: R N a Polymerase Levels And Calculated Rates Of Transcriptiomentioning

confidence: 99%

Quantification of Hepatic Transcribing RNA Polymerase Molecules, Polyribonucleotide Elongation Rates and Messenger RNA Complexity in Fed and Fasted Rats

1978

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We have investigated the mechanisms by which the rate of preribosomal RNA and premessenger RNA synthesis are repressed during starvation of rats by a quantitative study of the transcriptional events in isolated nuclei and by complexity analysis of the cytoplasmic polyadenylated RNA. The transcriptional activities of form A and B DNA-dependent RNA polymerases, which produce preribosomal and premessenger RNA respectively, were distinguished by the use of a-amanitin. Form C activity was shown to be absent from nuclei as isolated here.Polyribonucleotide elongation rates and the number of transcribing RNA polymerase molecules per diploid genome were determined by UMP/uridine analysis of the alkali-digested, nascent RNA labelled from r3H]UTP during 1-min incubations at 22 "C in the presence and absence of a-amanitin. The inhibition of form-A-mediated preribosomal RNA synthesis in response to starvation and cycloheximide treatment could be entirely accounted for by a decrease in the polyribonucleotide elongation rate. No significant change in the number of transcribing enzyme molecules (about 20 x lo3 per diploid genome) was detected. However on refeeding, the elongation rate was restored to normal and the number of transcribing form A molecules was elevated over the normal level. These results suggest that there are at least two modes of control acting on preribosomal RNA synthesis, one linked to protein synthesis and affecting the elongation rate, while a second regulates the number of form A RNA polymerase molecules transcribing the template.Form-B-mediated premessenger RNA synthesis responded to starvation by a decrease in the number of transcribing enzyme molecules from approximately 7 x lo3 to 3 x lo3 per diploid genome. This change was reversed on refeeding. Cycloheximide treatment did not affect premessenger RNA synthesis either via elongation or the number of transcribing enzyme molecules. The data indicate that premessenger RNA synthesis is controlled in this system at the level of RNA polymerase initiation by a mechanism not linked to protein synthesis.Hybridization analysis of the complexity of liver cytoplasmic polyadenylated mRNA from normal, starved and refed animals showed that three or four abundance classes were present in each case. No significant difference in mRNA populations between the dietary conditions was detected. More detailed examination of the most abundant class of mRNA confirmed this conclusion. The data show that although the number of transcribing form B molecules is reduced during starvation, the complexity and relative abundance of the mRNA synthesised does not change. The simplest explanation of these results is that, whilst the number of active genes does not change significantly during starvation, on average there are fewer form B enzyme molecules transcribing each gene in the starved animal.When rats are deprived of food there follows a progressive decrease in the RNA content of the liver [l -31. The activity of the nucleolar form A DNA-E~~~~~, RNA polymerase or nucleosidetriphos...

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Section: R N a Polymerase Levels And Calculated Rates Of Transcriptiomentioning

confidence: 99%

Quantification of Hepatic Transcribing RNA Polymerase Molecules, Polyribonucleotide Elongation Rates and Messenger RNA Complexity in Fed and Fasted Rats

1978

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“…In fact, most of this hnRNA turns over very rapidly in the nucleus, a half-life of 23 min has been reported in L cells [4], and only a small percentage ever reaches the cytoplasm [4,5].…”

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confidence: 99%

Characterisation of an Endogenous Protein Kinase Activity in Ribonucleoprotein Structures Containing Heterogenous Nuclear RNA in HeLa Cell Nuclei

Blanchard

Brunel²,

Jeanteur³

1977

European Journal of Biochemistry

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Ribonucleoprotein particles containing heterogeneous nuclear RNA (hnRNA) were isolated from sonically disrupted HeLa cells nuclei by sedimentation through a triple sucrose cushion. We found that these particles were able to catalyse the incorporation of radioactive phosphate from [Y-~~PIATP into phosphoserine and phosphothreonine residues of endogenous proteins. Optimal conditions for enzymatic activity were defined after investigation of the influence of several parameters of the reaction. The requirement for a divalent cation was most efficiently met by 10 mM Mg', to a lower extent by Mn2+ and not at all by Ca2'. An apparent K, value for ATP of 0.02 mM was determined and the enzyme was found to be sensitive to p-hydroxymercuribenzoate. Neither cyclic AMP nor cyclic GMP stimulated the reaction over a wide range of concentrations and the same protein substrates were phosphorylated in their presence.After excluding possible contamination by other subcellular fractions, considerable evidence for the association of kinase and phosphate acceptor proteins with ribonucleoprotein structures containing hnRNA was gathered along several lines by showing that the enzymatic activity closely followed tritiated RNA pulse-labeled in the presence of low doses of actinomycin D in the following circumstances : (a) sedimentation in sucrose gradients, (b) isopycnic banding in metrizamide density gradients in the presence or absence of M g ' ions and (c) adsorption to oligo(dT)-cellulose columns under conditions where poly(A)-containing material is adsorbed.Analysis of phosphorylated substrates by electrophoresis on polyacrylamide gels containing sodium dodecylsulphate revealed that, although they spanned a wide range of molecular weights, the most intensely labeled species were localized in bands corresponding to molecular weights of 37000 and 28 000.The above pattern of polypeptides phosphorylated in vitro is compared with that of phosphoproteins labeled in vivo after exposure of cells to [32P]phosphate.The most widely accepted model for messenger RNA formation in eucaryotic cells postulates that it is derived by post-transcriptional modification of the rapidly labeled heterogeneous nuclear RNA (hnRNA, for review see [l]). However, only in few instances could a direct precursor-product relationship between this hnRNA and cytoplasmic messenger RNA be established with good evidence [2,3].Abbreviations. hnRNA, heterogenous nuclear RNA; mRNA, messenger RNA; hnRNA . proteins, heterogenous nuclear ribonucleoprotein particles; CAMP and cGMP, adenosine and guanosine 3': 5'-monophosphates.

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“…to think that RNA obtained from the nuclear preparations was really of nuclear origin in spite of its unusually high yield. It is clearly seen ( fig.1) that the optical density profile of nuclear RNA resembles that of one typical of a polysomal RNA containing two sufficiently homogeneous peaks: 18 S and 28 S. The 18 S peak could be identified partly as the bulk of nuclear messenger RNA [16] and in part as a cytoplasmic contaminant (see [2,6]). We were concerned with the reasons for the presence in the nuclei of such a great amount of a homogeneous 28 S RNA and about its origin.…”

Section: The Content Sedimentation Properties and Metabolic Stabilitmentioning

confidence: 99%

“…This RNA class is extremely polydisperse in molecular weight and metabolically unstable its half-life does not exceed 30 mm [2,4,5] . This presumes that in about 3 h not more than 1% of the newly formed HnRNA should remain in the nuclei.…”

Section: Introductionmentioning

confidence: 99%

“…As shown in this work, for example, at least 35% of the total RNA is found in the nuclei of pigeon-bone marrow cells. It is also a puzzling point that the sedimentation profile of the optically measured nuclear RNA looks more like that of ribosomal RNA [2, than of HnRNA. The suggestion that the bulk of nuclear RNA is represented only by extremely unstable HnRNA and pre-rRNA [2] seems hardly probable.…”

Section: Introductionmentioning

confidence: 99%

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Metabolically stable messenger‐like 28 S RNA fraction in the nuclei of pigeon‐bone marrow cells

et al. 1977

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Stability of nuclear RNA in mammalian cells

Cited by 193 publications

References 32 publications

Quantification of Hepatic Transcribing RNA Polymerase Molecules, Polyribonucleotide Elongation Rates and Messenger RNA Complexity in Fed and Fasted Rats

Quantification of Hepatic Transcribing RNA Polymerase Molecules, Polyribonucleotide Elongation Rates and Messenger RNA Complexity in Fed and Fasted Rats

Characterisation of an Endogenous Protein Kinase Activity in Ribonucleoprotein Structures Containing Heterogenous Nuclear RNA in HeLa Cell Nuclei

Metabolically stable messenger‐like 28 S RNA fraction in the nuclei of pigeon‐bone marrow cells

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