Stimulation of Polypeptide Initiation
            <i>In Vitro</i>
            After Protein Synthesis Inhibition
            <i>In Vivo</i>
            in HeLa Cells

Reichman, Marsha E.; Penman, Sheldon

doi:10.1073/pnas.70.9.2678

Cited by 21 publications

(11 citation statements)

References 27 publications

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“…More than 20 years ago, Reichman and Penman identified a factor, termed an activator, which stimulates translational initiation (37). They demonstrated that this activator is an RNA with a half-life of about 1 h, which is approximately the short lifetime of flAlu RNA (6,16).…”

Section: Discussionmentioning

confidence: 99%

Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR

Chu

Ballard

Carpick

et al. 1998

Molecular and Cellular Biology

182

147

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Cell stress, viral infection, and translational inhibition increase the abundance of human Alu RNA, suggesting that the level of these transcripts is sensitive to the translational state of the cell. To determine whether Alu RNA functions in translational homeostasis, we investigated its role in the regulation of double-stranded RNA-activated kinase PKR. We found that overexpression of Alu RNA by cotransient transfection increased the expression of a reporter construct, which is consistent with an inhibitory effect on PKR. Alu RNA formed stable, discrete complexes with PKR in vitro, bound PKR in vivo, and antagonized PKR activation both in vitro and in vivo. Alu RNAs produced by either overexpression or exposure of cells to heat shock bound PKR, whereas transiently overexpressed Alu RNA antagonized virus-induced activation of PKR in vivo. Cycloheximide treatment of cells decreased PKR activity, coincident with an increase in Alu RNA. These observations suggest that the increased levels of Alu RNAs caused by cellular exposure to different stresses regulate protein synthesis by antagonizing PKR activation. This provides a functional role for mammalian short interspersed elements, prototypical junk DNA.

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Section: Discussionmentioning

confidence: 99%

Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR

Chu

Ballard

Carpick

et al. 1998

Molecular and Cellular Biology

182

147

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“…Previous evidence from both in vivo studies and in vitro ability to initiate protein synthesis has indicated that some metabolic activity involving RNA is necessary to maintain proper initiation rates in HeLa cells. This process is inhibited by actinomycin (2,10,11). When this phenomenon was examined in detail in in vivo experiments, it was found that the actinomycin concentration required for half-maximum effect was 0.1-0.2 ,g/ml.…”

Section: Resultsmentioning

confidence: 99%

“…Several lines of experimentation suggest that an important site of control is the process of initiation (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). In exponentially growing HeLa cells, it has been possible to induce enhanced rates of initiation in vivo (2,10) by exposing cells to a period of decreased protein synthesis caused by elevated temperature, treatment with cycloheximide, or starvation for an essential amino acid.…”

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confidence: 99%

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The Regulation of Protein Synthesis in Mammalian Cells VI. Soluble and Polyribosome Associated Components Controlling In Vitro Polypeptide Initiation in HeLa Cells

Goldstein

Reichman

Penman

1974

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

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The in vitro initiation of polypeptides on endogenous polyribosomes has been studied in extracts from HeLa cells. Regulation of the rate of initiation of polypeptides can be examined. In these experiments an assay using [uSjfMet-tRNAiMet has been developed, and the system further characterized.The system has been separated into a fraction containing polyribosomes with subunits and a fraction containing soluble components. The regulation of initiation has at least two distinct components.There is one factor in the soluble fraction which develops a stimulated response after protein synthesis has been in. hibited in intact cells. This stimulation does not require new RNA synthesis during the period of cell "stress."A second component is associated with ribosomes. This factor is necessary for the initiation of polypeptides on endogenous polyribosomes. It disappears gradually when cells are exposed to actinomycin. The disappearance is first manifested by an inability of polyribosomes to respond to stimulated supernatants. This unstable component, which decays in the presence of actinomycin, has no apparent counterpart in systems that measure initiation on exogenous mRNA.The control of the translation of a relatively stable population of messenger RNA molecules appears to be a significant aspect of regulation of protein synthesis in mammalian cells. Several lines of experimentation suggest that an important site of control is the process of initiation (1-12). In exponentially growing HeLa cells, it has been possible to induce enhanced rates of initiation in vivo (2, 10) by exposing cells to a period of decreased protein synthesis caused by elevated temperature, treatment with cycloheximide, or starvation for an essential amino acid. We shall refer to cells that have been exposed to conditions inhibiting protein synthesis as "stressed." We shall show that stressing cells results directly in activating a soluble factor in the cytoplasm which leads to enhanced rates of initiation.A second distinct phenomenon is the gradual loss of the capacity of polyribosomes to initiate polypeptides both in vivo and in vitro when new RNA synthesis is blocked by actinomycin. The lesion occurs prior to the loss of significant amounts of mRNA and appears due to a block by the drug of the appearance of a component necessary for proper initiation on polyribosomes. Furthermore,-the loss of this factor in actinomycin during "stress" prevents polyribosomes from responding to the activated soluble fraction. We believe this ribosome-associated factor can be demonstrated only on endogenous polyribosomes from cells with active RNA metabolism (Reichman, Goldstein, and Penman, manuscript in preparation).In order to study the biochemical events involved in the cellular control of the initiation of translation, an in vitro system reflecting the in vivo state was needed. Recently, such a system has been developed using HeLa cells (11

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“…Earlier the involvement of a small RNA in protein biosynthesis was postulated by several authors (2)(3)(4). Lowering of the level of this RNA might be responsible for the observed decline in protein synthesis after the administration of actinomycin D (3).…”

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confidence: 99%

Non-specific stimulation of cell-free protein synthesis by a dialyzable factor isolated from reticulocyte initiation factors ("iRNA").

Berns¹,

Salden²,

Bogdanovsky³

et al. 1975

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

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The specificity of a dialyzable component, isolated from rabbit reticulocyte initiation factors, in stimulating protein synthesis was examined. It appeared that this factor (hereafter designated as "iRNA") was able to restore the activity of initiation factor preparations that were inactivated by dialysis. The "iRNA" from reticulocytes stimulated polypeptide synthesis directed by the homologous globin mRNA as well as heterologous lens crystallin mRNAs. No selectivity in its stimulating action with regard to the type of mRNA studied cpuld be observed.The source of ribosomes or supernatant enzymes did not influence the effect of "iRNA". However, in an ascites lysate that was dependent on the addition of initiation factors, "iRNA" increased polypeptide formation only marginally, suggesting that in this lysate a similar factor was glready present in an active form. It is concluded that "iRNA" may regulate protein synthesis, but without exhibiting specificity towards mRNAs, at least those tested so far.Recently Bogdanovsky et al. reported the isolation of a small RNA species that was able to restore the activity of a dialyzed initiation factor preparation (1). Earlier the involvement of a small RNA in protein biosynthesis was postulated by several authors (2-4). Lowering of the level of this RNA might be responsible for the observed decline in protein synthesis after the administration of actinomycin D (3). The assumption was made that this hypothetical RNA with a short half-life was involved in the regulation of protein synthesis at the translational level. It was tempting to speculate that an RNA of this type might play a role in the regulation of protein synthesis by discriminating between mRNAs during initiation. The reported specificity of initiation factor IF3 towards different mENAs (5-7) could well be explained by a mechanism in which an initiation factor bound RNA would interact specifically with a certain mRNA (1).We report here that the KCl-wash RNA ("iRNA") from reticulocyte ribosomes does not exhibit specificity towards messengers from different origins and that no specificity was found with respect to the ribosomes and supernatant factors used. Furthermore, we show that a factor with the same type of activity may be found on ribosomes of different Qrigin and that part of this activity is also present in the supernatant. MATERIALS AND METHODSIsolation of "iRNA" from reticulocytes Rabbit reticulocytes and ribosomes were prepared as described previously by Miller and Schweet (8). For preparation of crude initiation factors polyribosomes were suspended in 0.25 M sucrose, 0.1 M Tris HCI, pH 7.6, at a concentration of about 450 A260 units/ml, adjusted to 0.5 M KCl with 4 M KCl, and gently stirred for 2 hr at 00. The ribosomes were removed by centrifugation at 130,000 X g for 3 hr at 40. The 0.5 M KCl-wash, diluted to 50 A4o units/ml, was dialyzed for 5 hr at 40 against 10 volumes of distilled water. The dialysate was Iyophilized, dissolved in half the volume of the original factor preparation, and fro...

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Stimulation of Polypeptide Initiation In Vitro After Protein Synthesis Inhibition In Vivo in HeLa Cells

Cited by 21 publications

References 27 publications

Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR

Potential Alu Function: Regulation of the Activity of Double-Stranded RNA-Activated Kinase PKR

The Regulation of Protein Synthesis in Mammalian Cells VI. Soluble and Polyribosome Associated Components Controlling In Vitro Polypeptide Initiation in HeLa Cells

Non-specific stimulation of cell-free protein synthesis by a dialyzable factor isolated from reticulocyte initiation factors ("iRNA").

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