Production of mRNA in chinese hamster cells: Relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences

Harpold, Michael M.; Evans, Ronald M.; Salditt-Georgieff, M; Darnell, James

doi:10.1016/0092-8674(79)90341-6

Cited by 320 publications

(130 citation statements)

References 65 publications

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“…1B) which is not substantially greater than that of poly(A)+mRNA. Recent results on processing times for individual HeLa cell hnRNAs (32) suggest that these size differences most likely reflect rapid in vivo processing events. In any event, the average size of steady-state nuclear poly(A)+RNA is about that of polysomal total mRNA.…”

Section: Resultsmentioning

confidence: 99%

Nonadenylylated mRNA is present as polyadenylylated RNA in nuclei of Drosophila.

Zimmerman¹,

Fouts²,

Lévy³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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The sequence complexity of nuclear total RNA and nuclear poly(A)+RNA from Drosophilathird-instar larvae was determined by hybridization of these RNAs to labeled single-copy DNA. At saturation, the nuclear poly(A)+-and total RNA hybridized to 11% and 22.5% of the single-copy DNA, respectively. The increase in complexity ofnuclear total RNA over that observed for nuclear poly(A)+RNA indicates the presence of a discrete class of nonadenylylated nuclear RNA molecules. The relationship between DNA sequences coding for nuclear RNA and mRNA was then determined by hybridization of nuclear total and poly(A)+RNA to DNA enriched for mRNA coding sequences. The results ofthese studies show that those single-copy DNA sequences that are represented in either the poly(A)+-or poly(A)-mRNA population are transcribed into RNA molecules that appear in the nuclear poly(A)+RNA population.It has been proposed that high molecular weight nuclear RNA serves several functions within the eukaryotic cell, the most widely accepted ofwhich is as a precursor to cytoplasmic mRNA (1). Although little direct evidence for this role existed for several years, recent advances in molecular techniques have resulted in the identification and characterization ofnuclear precursors for several specific mRNAs (2-5). In these studies, it has been clear that the mRNA sequences represent only a portion of the primary transcripts, and hence specific processing appears to be an essential feature and potential regulatory event in mRNA biogenesis. In lower eukaryotes (i.e., fungi) there is little or no difference in either the complexity or the size of the RNAs found in the nucleus and the cytoplasm (6, 7); in these systems the relationship between nuclear transcripts and translated mRNAs appears to be direct. In higher eukaryotes, where nuclear RNA is several times more complex than mRNA, the relationship between the nuclear and mRNA species is not as clearly understood. In mouse and rat brain (8, 9), RNA-DNA hybridization experiments clearly show that the polyadenylylated nuclear RNA population contains all of the polyadenylylated cytoplasmic RNA sequences. However, it is also clear that both the nuclear RNA and mRNA are composed of poly(A)+-and poly(A)-RNA populations (8)(9)(10). At present, the relationship between the nonadenylylated nuclear RNA and mRNA in these systems is unknown.In Drosophila, however, a recent observation by Lengyel et al. (11) clearly indicates that the transcribed region ofone ofthe major Drosophila heat shock puff sites, 93D, is represented almost exclusively by poly(A)-RNA in the cytoplasm, whereas it is represented by both poly(A)+-and poly(A)-RNA in the nucleus.In view ofthis fact, and because we have determined that the mRNA of Drosophila third-instar larvae is composed of both adenylylated and nonadenylylated species (12), we have measured the sequence complexity of poly(A)+-and total nuclear RNA present in these larvae. We report here that, as was observed in larval mRNA, the nuclear RNA is composed of both an adenyly...

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Section: Resultsmentioning

confidence: 99%

Nonadenylylated mRNA is present as polyadenylylated RNA in nuclei of Drosophila.

Zimmerman¹,

Fouts²,

Lévy³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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“…5B). The complex that forms between Sertoli cell nuclear proteins and the wtAP1 probe forms at the AP1/CRE-like site within the (Harpold et al, 1979). man TFIID antibody was unable to supershift the specific, nor the nonspecific complexes (Fig.…”

Section: Inducible C-fos Complex Formation On the Ap1/cre-like Sitementioning

confidence: 99%

Mechanisms involved in the homologous down-regulation of transcription of the follicle-stimulating hormone receptor gene in Sertoli cells

Griswold

Kim

Tribley

2001

Molecular and Cellular Endocrinology

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The action of follicle-stimulating hormone (FSH) in spermatogenesis is regulated at a fundamental level by controlling the number of competent receptors present at the surface of Sertoli cells. By controlling the number of receptors, the cell is able to modulate the timing and magnitude of subsequent signal transduction in response to FSH. One mechanism of control is the down-regulation of the steady state levels of the FSH receptor gene after exposure to FSH or agents that stimulate or prolong the cAMP signal transduction cascade (homologous down-regulation) in Sertoli cells. The goals of this study were to examine possible mechanisms involved in the down-regulation of mRNA levels of this gene. Analysis of transcription and processing by a PCR-based assay showed that treatment of Sertoli cells with FSH caused at least a 50% reduction of hnRNA for the FSH receptor gene. Reporter genes controlled by 5% flanking sequences of the FSH receptor gene that were transiently transfected into Sertoli cells were not down-regulated. In electrophoretic mobility shift assays (EMSA), cAMP-inducible nuclear protein complex containing c-Fos formed on the activator protein-1/cAMP responsive element-like site located at − 216 to − 210 in the promoter of the rat FSH receptor gene. We concluded from this study that there was no evidence for the putative role of ICER in the down-regulation of the FSH receptor promoter. In addition, the FSH-induced down-regulation of the transcription of the FSH receptor gene in Sertoli cells was prevented by the treatment of Sertoli cells with trichostatin A prior to the addition of FSH. This experiment coupled with other observations suggested that the down-regulation may be mediated by changes in chromatin structure.

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“…At indicated times cells were fractionated into nuclear and cytoplasmic fractions and the poly(A)c ytoplasmic RNA, prepared as in Harpold et al . (18) . A portion of each sample was subjected to complete T1 RNAse digestion and poly(U)-Sepharose chromatography to collect poly(A) for gel electrophoretic analysis (19) .…”

Section: Effects Of Actinomycin On Poly(a) Synthesismentioning

confidence: 99%

Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

Salditt-Georgieff¹,

Harpold²,

Sawicki³

et al. 1980

The Journal of Cell Biology

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A kinetic analysis of the appearance of [3H]uridine label in RNA sequences that neighbor poly(A), as well as the incorporation of [3H]adenosine label into both the RNA chain and the poly(A) of poly(A)-containing molecules, shows that poly(A) is added within a minute or so after RNA chain synthesis in Chinese hamster ovary cells and HeLa cells. Previous conclusions by several groups (5-7) that poly(A) might be added as long as 20-30 min after RNA synthesis appear to be in error, and the present conclusion seems much more in line with several different types of recent studies with specific mRNAs that suggest prompt poly(A) addition (13-16).

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Production of mRNA in chinese hamster cells: Relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences

Cited by 320 publications

References 65 publications

Nonadenylylated mRNA is present as polyadenylylated RNA in nuclei of Drosophila.

Nonadenylylated mRNA is present as polyadenylylated RNA in nuclei of Drosophila.

Mechanisms involved in the homologous down-regulation of transcription of the follicle-stimulating hormone receptor gene in Sertoli cells

Addition of poly(A) to nuclear RNA occurs soon after RNA synthesis.

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