Occurrence of Uridylate-Rich Oligonucleotide Regions in Heterogeneous Nuclear RNA of HeLa Cells

Molloy, George R.; Thomas, William; Darnell, James

doi:10.1073/pnas.69.12.3684

Cited by 61 publications

(18 citation statements)

References 34 publications

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“…Of interest to these observations is the finding by Molloy et al (11) concerning the occurrence of uridylate-rich regions in heterogeneous nuclear RNA of HeLa cells. These regions, consisting of approximately 30 nucleotides, appear to be absent in mRNA and are not located at the 3' end of heterogeneous nuclear RNA.…”

Section: Methodsmentioning

confidence: 93%

“…At the 3' end, the mRNA is located which is subsequently adenylylated. This aspect of the model is based on the findings of Molloy et al (11). The size of the poly(U) tract reported by Molloy et al (11), is within size range of the poly (U)-[3H]poly(A) hybrids we report from the mRNP-tcRNA.…”

Section: Methodsmentioning

confidence: 99%

“…This aspect of the model is based on the findings of Molloy et al (11). The size of the poly(U) tract reported by Molloy et al (11), is within size range of the poly (U)-[3H]poly(A) hybrids we report from the mRNP-tcRNA. The proposed model, suggests that on the 3' end of the poly(U) tract a sequence of nucleotides exists that will specifically recognize a sequence of nucleotides at the 5' end of the mRNA.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Two classes of translational control RNA: their role in the regulation of protein synthesis.

Bester¹,

Kennedy²,

Heywood³

1975

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

104

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Two classes of translational control RNA (tcRNA) have been isolated from embryonic chick muscle. One of these classes, the tcRNA isolated from messenger ribonucleoprotein particles (mRNP-tcRNA), is effective in inhibiting the translation of mRNP-mRNA while having little if any effect on polysomal mRNA. The other class, polysome-tcRNA, has no effect on mRNP-mRNA while it stimulates the translation of polysomal mRNA. Translational control RNA (tcRNA) has been isolated from initiation factor preparations and has been demonstrated to specifically inhibit the translation of heterologous mRNAs (3). The inhibition by muscle tcRNA results from the inability of the globin mRNA to bind to ribosomes during the formation of the initiation complex when tcRNA is added to the cell-free system (6). Due to the specific nature of this inhibition, it is likely that the tcRNA interacts directly with mRNA. However, the involvement of initiation factors in this tcRNA-mediated inhibition cannot be excluded.In order to determine if tcRNA is effective in the regulation of protein synthesis within the cell from which it is derived, we have isolated mRNA from both polysomes and messenger ribonucleoprotein particles (mRNPs) of embryonic chick muscles and tested the ability of muscle tcRNA to inhibit the translation of these mRNAs. We report here that tcRNA may be obtained from either the polysomal or mRNP sources. Both tcRNA preparations inhibit globin synthesis, while only the tcRNA obtained from mRNPs is effective in inhibiting the Abbreviations: tcRNA, translational control RNA; mINP, messenger ribonucleoprotein particles. * Permanent address: Tygerberg Hospital, Capetown, South Africa. 1523translation of muscle mRNAs; and, in this case, only the translation of mRNP-mRNA is inhibited. It therefore is likely that at least two classes of tcRNA are present in muscle. It was of particular interest that the tcRNA effective in inhibiting muscle mRNP-mRNA translation contains a poly(U) tract that is absent in the polysomal class of tcRNA. From these results, a model is proposed to explain the manner by which tcRNA specifically regulates the utilization of cytoplasmic mRNAs. MATERIALS AND METHODSThe preparation of rabbit reticulocyte lysates and the conditions for cell-free protein synthesis were as described (3) except that 50 AM hemin and 15 jAg of muscle initiation factor 3 (IF3) were present in the reaction mixture, which had a final volume of 0.1 ml. Incubations were for 30 min at 300. Twentyfive one hundredth nmol each of 20 amino acids containing 8 jACi of 3H-labeled amino-acid mixture (New England Nuclear Corp.) was added to each incubation mixture. Where indicated, 2 ,g of tcRNA and 15 ug of poly(A)-containing mRNA from muscle were added to the cell-free, amino-acid incorporating systems.The mRNA and tcRNA preparations were prepared from 14-day embryonic chick leg muscle in the following manner. Approximately 45-50 g of muscle was gently homogenized in 0.25 M KC1, 5 MM M\gCI2, 20 mM Tris-HCl (pH 7.4) as described (7). The 10,00...

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Section: Methodsmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Two classes of translational control RNA: their role in the regulation of protein synthesis.

Bester¹,

Kennedy²,

Heywood³

1975

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

104

View full text Add to dashboard Cite

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“…The presence of short oligo(U) sequences of about 30 nucleotides in hnRNA was first demonstrated by Molloy and coworkers (64) and shown not to be localized near the 3'terminus (65) although this point is still in dispute (22). There are indications that these sequences are present only in polyadenylated hnRNA and as such may contribute to the secondary structure of hnRNA (66).…”

Section: The Presence Of Oligo(u) Sequencesmentioning

confidence: 99%

HnRNP particles

Venrooij¹,

Janssen²

1978

Mol Biol Rep

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“…Small amounts of longer polynucleotides have been detected but not defined. Because relatively long polyadenylate segments (6)(7)(8)(9)(10)(11) and uridylate-rich regions (12) have been found in cellular RNA, we have examined pyrimidine polynucleotides in HeLa cell nuclear DNA to see if corresponding sequences could be detected. Pyrimidine polynucleotides were separated according to chain length by polyacrylamide gel electrophoresis and, unexpectedly, a large amount of long polynucleotides was found.…”

mentioning

confidence: 99%

Analysis of Long Pyrimidine Polynucleotides in HeLa Cell Nuclear DNA: Absence of Polydeoxythymidylate

Birnboim

Mitchel

Straus

1973

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

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HeLa cell nuclear DNA contains an unexpectedly large amount of long pyrimidine polynucleotides.These sequences were detected in DNA labeled with ['HI thymidine by treatment with formic acid-diphenylamine and subsequent analysis by polyacrylamide gel electrophoresis. About 0.5% of the total thymine residues were found in polynucleotides that migrated more slowly than 4S RNA. No polynucleotides of comparable size were detected in Escherichia coli DNA. The pyrimidine polynucleotides contained no pure poly(dT) sequences (less than 0.0015% of the total residues), as judged by thermal chromatography of a complex formed with poly(A).When high molecular weight DNA is treated with acid under relatively mild conditions (1, 2), purine bases are selectively removed, the phosphodiester backbone is broken, and pyrimidine clusters having the general formula, p(Pyp)5, are released. The largest pyrimidine oligonucleotides previously isolated from animal cell DNA contain 15-20 nucleotides (3)(4)(5). Small amounts of longer polynucleotides have been detected but not defined. Because relatively long polyadenylate segments (6-11) and uridylate-rich regions (12) have been found in cellular RNA, we have examined pyrimidine polynucleotides in HeLa cell nuclear DNA to see if corresponding sequences could be detected. Pyrimidine polynucleotides were separated according to chain length by polyacrylamide gel electrophoresis and, unexpectedly, a large amount of long polynucleotides was found. Since their size and amount differ very greatly from that predicted assuming a random distribution of pyrimidines, they may represent sequences of special significance in the genome. When annealed with commercial poly(A), almost no pure poly(dT) sequences could be detected. MATERIALS AND METHODSPreparation of ['HIDNA. HeLa cells used in these experiments were grown in spinner culture in Eagle's minimal essential medium with 5% calf serum without antibiotics. 400 ml of cell suspension (3 X 105/ml) were incubated with [methyl-'H ]thymidine at a concentration of 1 gCi/ml, 0.5 AM, for 24 hr. Nuclei were isolated as described by Penman (13); the nuclear pellet was suspended in 0.5 ml of hypotonic buffer (10 mM NaCl-10 mM Tris HCl-3 mM MgCl2) and rapidly mixed with 8 ml of lysing solution [1 M LiCl, 0.1 M EDTA, 0.1 M Tris HCl (pH 8.0), and 1% Na dodecyl sulfate]. The suspension was heated at 600 for 5 min, then cooled to room temperature and passed twice through a no. 26 needle to reduce its viscosity. The preparation was deproteinized by shaking at room temperature for 30 min with an equal 2189 volume of chloroform containing 0.1 ml n-octanol. The aqueous phase was recovered by centrifugation and the procedure was repeated three times more. DNA was precipitated with ethanol and dissolved in 2 ml of a solution containing 10 mM NaCl, 1 mM EDTA, 1 mM Tris -HCl (pH 7.4), and 0.2% Na dodecyl sulfate. NaOH was added to 0.15 N, and the DNA was heated at 600 for 10 min to hydrolyze RNA. The sample was neutralized with 3 N acetic acid, and the DNA was pre...

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Occurrence of Uridylate-Rich Oligonucleotide Regions in Heterogeneous Nuclear RNA of HeLa Cells

Cited by 61 publications

References 34 publications

Two classes of translational control RNA: their role in the regulation of protein synthesis.

Two classes of translational control RNA: their role in the regulation of protein synthesis.

HnRNP particles

Analysis of Long Pyrimidine Polynucleotides in HeLa Cell Nuclear DNA: Absence of Polydeoxythymidylate

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