Relationship of chain transcription to poly(A) addition and processing of hnRNA in HeLa cells

Derman, E; Darnell, James

doi:10.1016/0092-8674(74)90140-8

Cited by 92 publications

(36 citation statements)

References 26 publications

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“…DISCUSSION Our kinetic studies of poly(A) metabolism in L cells show that nuclear poly(A) accumulates to steady state with a halftime of approximately 25 min, similar to that of hnRNA (6), and that radioactivity in cytoplasmic poly(A) begins to accumulate with little or no lag. These data are consistent with a variety of complex models that describe the relationship beall the critical parameters can be measured independently using current technology; therefore our results, like those reported previously (3,4,17,20), do not lead to a unique interpretation. However, we can now restrict the range of models that are consistent with the data.…”

Section: Figuresupporting

confidence: 79%

“…This suggests that there may be more than one class of polyadenylated nuclear RNA molecules, in agreement with other observations (20,30). Since there is a lag in attainment of a maximum rate of accumulation of polyadenylated mRNA in the cytoplasm (4,31), this poly(A) would be attached to precursors of mRNA just before they pass into the cytoplasm but after they had resided in the nucleus an average of several minutes.…”

Section: Figuresupporting

confidence: 75%

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Relationship between nuclear and cytoplasmic poly(adenylic acid).

Brandhorst¹,

McConkey²

1975

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

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The kinetics of accumulation of radioactive poly(A) in the nucleus and cytoplasm of mouse L cells have been determined using labeling conditions in which the cellular ATP pool is shown to have a nearly constant specific radioactivity. Most or all nuclear poly(A) accumulates with kinetics very similar to those of heterogeneous nuclear RNA, having a half-time of about 25 min. There is little or no lag before attainment of a maximal rate of accumulation of cytoplasmic poly(A). These data are consistent with a variety of models in which nuclear poly(A) does or does not all serve as a precursor of cytoplasmic poly(A). In either type of model there must be a class of poly(A) either synthesized in the cytoplasm or passing through a small nuclear pool separate from the main pool of nuclear poly(A). The T3' ends of many messenger RNA (mRNA) and heterogeneous nuclear RNA (hnRNA) molecules are polyadenylated posttranscriptionally. This observation has led to the suggestion that polyadenylated hnRNA molecules serve as precursors of mRNA (1). If the 3' ends of all polyadenylated hnRNA molecules were precursors of mRNA, these nuclear precursors could easily be selected from total hnRNA by affinity chromatography (2). If some polyadenylated hnRNA molecules decay entirely in the nucleus, such selection would not serve to isolate only precursors of mRNA. In the latter instance, some poly(A) would be expected to decay in the nucleus.Jelinek et al. (3) have presented evidence that most or all cytoplasmic poly(A) is derived from nuclear poly(A), and suggest that nuclear poly(A) may be conserved. More recently Perry, Kelley, and LaTorre (4) reported on their investigations of the kinetics of accumulation of poly(A) in the nucleus and cytoplasm of L cells and concluded that some nuclear poly(A) must decay in the nucleus and that substantial synthesis of cytoplasmic poly(A) may occur. Our investigations of ATP pools and nuclear RNA in L cells (5, 6) gave us reason to think that there might be sources of error in the investigations of Perry et al. (4). Perry et al. demonstrated that total cellular RNA labeled with tritiated adenosine at high concentration accumulates with kinetics indicating that it is synthesized via an ATP pool of constant specific radioactivity. Whereas, this appears to be valid over the long term, their argument cannot be convincingly applied over the first few hours, because it does not take into account the rapid initial accumulation of highly unstable hnRNA and nuclear precursors of ribosomal RNA, nor the extensive conversion of adenine nutleotides to guanine nucleotides under the labeling conditions used (5, 6). In the investigations of Perry et al. (4) the cellular ATP pool would not reach a constant specific radioactivity until as late as 2 hr after the addition of exogenous label (Brandhorst, unpublished observations); this could seriously distort the actual kinetics of accumulation of poly(A). As explained previously (4, 6), rapid establishment of constant specific activity of the ATP pool facilita...

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Section: Figuresupporting

confidence: 79%

Section: Figuresupporting

confidence: 75%

Relationship between nuclear and cytoplasmic poly(adenylic acid).

Brandhorst¹,

McConkey²

1975

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

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“…Using cloned immunoglobulin light chain recombinant plasmid DNA, we have detected discrete classes of rapidly labeled nuclear RNA containing K mRNA sequences which, after stringent denaturation (25,26), remain substantially larger than the K light chain mRNA in immunoglobulin-producing mouse myeloma cells. Kinetic labeling studies indicate that immunoglobulin K light chain mRNA appears to be processed from a large nuclear RNA species, approximately 10 times the size of K mRNA, through a nuclear RNA intermediate 3-4 times larger than K light chain mRNA and a nuclear RNA species similar in size to K mRNA.…”

mentioning

confidence: 99%

Immunoglobulin light chain mRNA is processed from large nuclear RNA.

Gilmore-Hebert¹,

Wall²

1978

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

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Recombinant DNA probes, produced by the molecular cloning of immunoglobulin K light chain mRNA, have been used to analyze heterogeneous nuclear RNA for presumptive precursors to cytoplasmic K light chain mRNA. Three discrete classes of nuclear RNA containing K mRNA sequences were detected after pulse-labeling of immunoglobulin-producing P3 myeloma cells. Two of these were substantially larger than K mRNA (approximately 10 and 4 times larger); the third was similar in size to K mRNA. Beginning with the largest, the sequential appearance of these three classes of nuclear RNA preceded the first a pearance of newly synthesized K light chain mRNA in the cytoplasm. The results presented here suggest that immunoglobulin K light chain mRNA is generated by the stepwise cleavage and processing of a large nuclear RNA transcript. Resolution of the events in the biogenesis of mRNA is essential to an understanding of the regulation of gene expression in eukaryotic cells. It is now well established that most mRNAs in eukaryotic cells are derived from nuclear RNA through post-transcriptional modifications including poly(A) addition (1-3), methylation, and the addition of 5'-terminal "caps" of the general structure m7G(5')pppN'-m-N"(m)p (4-6). Considerable evidence also suggests that the mRNA in eukaryotic cells is generated by the post-transcriptional cleavage of larger nuclear RNA (2,3,6). Compelling results now indicate that viral mRNA from the DNA tumor viruses simian virus 40 (7) and adenovirus (8-11) and -the cellular mRNA for globin (12)(13)(14)(15) are processed from substantially larger nuclear RNA. However, all mRNA in eukaryotic cells may not be processed from large nuclear RNA. RNA molecules larger than the respective cytoplasmic mRNA were not detected for ovalbumin mRNA (16) and murine RNA tumor virus RNA (17). However, short-lived nuclear RNAs, particularly those only marginally larger than their respective cytoplasmic mRNAs, may not be detected under the labeling and hybridization conditions generally used. In the case of silk fibroin mRNA, pulse-labeling at reduced temperature (presumably lowering the rate of RNA cleavage) was necessary to reveal a short-lived presumptive precursor only slightly larger than the silk fibroin mRNA (18)(19)(20) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. K light chain mRNA* by procedures previously established for the preparation of recombinant DNA clones from rabbit globin mRNA (22). Using cloned immunoglobulin light chain recombinant plasmid DNA, we have detected discrete classes of rapidly labeled nuclear RNA containing K mRNA sequences which, after stringent denaturation (25, 26), remain substantially larger than the K light chain mRNA in immunoglobulin-producing mouse myeloma cells. Kinetic labeling studies indicate that immunoglobulin K light chain mRNA appears to be processed from a large nuc...

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“…In the 18 hours 32p label experi-ments no actinomycin D was added and after 4 hours of labeling in phosphate-free medium cold inorganic phosphate was added to a concentration of 5xlO05M. To fractionate the RNAs by size both cytoplasmic and nuclear RNA fractions were made 95% in DMSO and then sedimented in sucrose gradients as described (7). The poly(A) sequence content and length were determined after digestion with Tl RNAse by poly(U) sepharose selection and acrylamide gel (15%) electrophoresis as described (4).…”

Section: Methodsmentioning

confidence: 99%