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

Salditt-Georgieff, M; Harpold, Michael M.; Sawicki, Stanley G.; Nevins, Joseph R.; Darnell, James

doi:10.1083/jcb.86.3.844

Cited by 46 publications

(29 citation statements)

References 32 publications

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“…Altogether, these early experiments provided very strong evidence (prior to the discovery of splicing) that much of the polyA was quickly added in the nucleus to molecules much larger than mRNA and that, beginning ∼5-10 min later, the same ∼230-nt 3 ′ polyA unit (Jelinek et al 1973;Sawicki et al 1977;Salditt-Georgieff et al 1980) began to accumulate in increasing amounts in the much smaller cytoplasmic polyribosomal mRNA. As we shall see, the early short-label experiments are consonant with the latest RNA seq studies on nascent, still chromatin-associated molecules (Bhatt et al 2012; A Pandya-Jones, DM Bhatt, C-H Lin, ST Smale, and DL Black, in prep.…”

Section: H Nucleosides and Pre-mrna Synthesis In Hela Cellsmentioning

confidence: 95%

“…Limited alkaline treatment of extracted nuclear nonribosomal RNA after brief label times of 3 H uridine incorporation (30 sec to 4 min) reduced all labeled molecules to an average size of ∼500 nt, and polyA + fragments and polyA − fragments were separated. The polyA − ∼500-nt hnRNA fragments and the polyA + fragments accumulated label at almost the same rate, with labeling of polyA+ fragments lagging by, at most, 30 sec (Salditt-Georgieff et al 1980). PolyA addition, therefore, occurred within, at most, 30 sec to newly synthesized nuclear RNA (Fig.…”

Section: H Nucleosides and Pre-mrna Synthesis In Hela Cellsmentioning

confidence: 99%

“…Cells were briefly labeled as indicated; total hnRNA was extracted, broken with brief alkali treatment to ∼500-nt fragments and polyA + and polyA − fragments counted (from Fig. 2 in Salditt-Georgieff et al 1980). cytoplasmic mRNA (see Puckett et al 1975), polyadenylation generally preceded the completion of processing, whatever that entailed.…”

Section: H Nucleosides and Pre-mrna Synthesis In Hela Cellsmentioning

confidence: 99%

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Reflections on the history of pre-mRNA processing and highlights of current knowledge: A unified picture

Darnell¹

2013

RNA

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Several strong conclusions emerge concerning pre-mRNA processing from both old and newer experiments. The RNAPII complex is involved with pre-mRNA processing through binding of processing proteins to the CTD (carboxyl terminal domain) of the largest RNAPII subunit. These interactions are necessary for efficient processing, but whether factor binding to the CTD and delivery to splicing sites is obligatory or facilitatory is unsettled. Capping, addition of an m 7 Gppp residue (cap) to the initial transcribed residue of a pre-mRNA, occurs within seconds. Splicing of pre-mRNA by spliceosomes at particular sites is most likely committed during transcription by the binding of initiating processing factors and ∼50% of the time is completed in mammalian cells before completion of the primary transcript. This fact has led to an outpouring in the literature about "cotranscriptional splicing." However splicing requires several minutes for completion and can take longer. The RNAPII complex moves through very long introns and also through regions dense with alternating exons and introns at an average rate of ∼3 kb per min and is, therefore, not likely detained at each splice site for more than a few seconds, if at all. Cleavage of the primary transcript at the 3 ′ end and polyadenylation occurs within 30 sec or less at recognized polyA sites, and the majority of newly polyadenylated pre-mRNA molecules are much larger than the average mRNA. Finally, it seems quite likely that the nascent RNA most often remains associated with the chromosomal locus being transcribed until processing is complete, possibly acquiring factors related to the transport of the new mRNA to the cytoplasm.

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Section: H Nucleosides and Pre-mrna Synthesis In Hela Cellsmentioning

confidence: 95%

Section: H Nucleosides and Pre-mrna Synthesis In Hela Cellsmentioning

confidence: 99%

Section: H Nucleosides and Pre-mrna Synthesis In Hela Cellsmentioning

confidence: 99%

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Reflections on the history of pre-mRNA processing and highlights of current knowledge: A unified picture

Darnell¹

2013

RNA

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“…After 12 to 15 min at 30°C (with occasional tapping), nuclei were pelleted, and nuclear RNA was extracted as described (23). RNA was precipitated twice from 8-ml samples with 2.5 volumes of ethanol, 0.2 M NaCl, and yeast tRNA carrier.…”

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confidence: 99%

Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes.

Clayton¹,

Darnell²

1983

Mol. Cell. Biol.

Self Cite

303

186

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Liver-specific mRNA sequences were examined in primary cultures of mouse hepatocytes. After cell disaggregation by collagenase treatment and for at least 24 h in culture, little change in liver-specific mRNA concentrations was noted. Gradually over a period of 140 h, liver-specific mRNAs declined. In contrast, transcriptional assays in which liver cell nuclei were used to produce 32P-labeled nuclear RNA showed that liver-specific gene transcription was greatly diminished within 24 h, while polymerase II transcription of "common" genes and transcription of tRNA and rRNA did not decline. Thus, a prompt differential transcriptional effect seems to underlie the gradual loss of tissue specificity of the primary cultures.

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“…But current information on polyadenylation rates in vivo is limited by the methodology available for making such measurements. One such method involves the measuring of the pulse-labelled RNA in the poly(A)+ fraction of total nuclear RNA (Salditt-Georgieff et al, 1980). Such method is inherently limited in precision and only suitable for RNA that is abundant.…”

Section: Introductionmentioning

confidence: 99%

Dual Luciferase Assay: an Efficient Method to Study the Polyadenylation Process

Jamil¹,

Yaqub²,

Sheikh³

et al. 2000

Pakistan J. of Biological Sciences

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Cleavage and polyadenylation is an obligatory step in mRNA biogenesis in eukaryotes. Poly(A) tails play an important role in mRNA turn over, transport and translation. Currently available methods for the study of polyadenylation are very cumbersome and involve radioactivity. Dual luciferase assay was found to be an efficient and equally reliable method to CAT assay and RNase protection assay. Comparison of these methods is reported in this paper.

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

Cited by 46 publications

References 32 publications

Reflections on the history of pre-mRNA processing and highlights of current knowledge: A unified picture

Reflections on the history of pre-mRNA processing and highlights of current knowledge: A unified picture

Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes.

Dual Luciferase Assay: an Efficient Method to Study the Polyadenylation Process

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