In vitro release of α1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo

Gary Clawson, J. Button, C. H. Woo, Yu Cheng Liao, E. A. Smuckler

Research output: Contribution to journalArticle

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Abstract

The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in α1-acid glycoprotein (αAGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron αAGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, αAGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A) + RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some αAGP sequences in smaller, more heterogeneous poly(A)-RNA, and leakage of some αAGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature αAGP mRNA to the nucleus. In contrast to αAGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest αAGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A) + RNA preparations which may represent 3′-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of αAGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of αAGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of αAGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of αAGP in the acute phase response.

Original languageEnglish (US)
Pages (from-to)163-172
Number of pages10
JournalMolecular Biology Reports
Volume11
Issue number3
DOIs
StatePublished - Sep 1 1986

Fingerprint

Acute-Phase Reaction
Glycoproteins
RNA
Acids
Messenger RNA
Introns
Turpentine
Povidone
Exons
Nuclear Envelope
In Vitro Techniques
Adenosine Triphosphate
RNA Transport
Membranes
Nuclear RNA
Cell Nucleus Active Transport
Ribonucleases
Detergents
Liver
Placenta

All Science Journal Classification (ASJC) codes

  • Genetics
  • Cell Biology
  • Biochemistry

Cite this

Clawson, Gary ; Button, J. ; Woo, C. H. ; Liao, Yu Cheng ; Smuckler, E. A. / In vitro release of α1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo. In: Molecular Biology Reports. 1986 ; Vol. 11, No. 3. pp. 163-172.
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abstract = "The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in α1-acid glycoprotein (αAGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron αAGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, αAGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A) + RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some αAGP sequences in smaller, more heterogeneous poly(A)-RNA, and leakage of some αAGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature αAGP mRNA to the nucleus. In contrast to αAGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest αAGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A) + RNA preparations which may represent 3′-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of αAGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of αAGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of αAGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of αAGP in the acute phase response.",
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In vitro release of α1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo. / Clawson, Gary; Button, J.; Woo, C. H.; Liao, Yu Cheng; Smuckler, E. A.

In: Molecular Biology Reports, Vol. 11, No. 3, 01.09.1986, p. 163-172.

Research output: Contribution to journalArticle

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AU - Button, J.

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N2 - The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in α1-acid glycoprotein (αAGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron αAGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, αAGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A) + RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some αAGP sequences in smaller, more heterogeneous poly(A)-RNA, and leakage of some αAGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature αAGP mRNA to the nucleus. In contrast to αAGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest αAGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A) + RNA preparations which may represent 3′-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of αAGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of αAGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of αAGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of αAGP in the acute phase response.

AB - The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in α1-acid glycoprotein (αAGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron αAGP sequences. Contamination of nuclear preparations by membrane-absorbed cytoplasmic RNA was eliminated by detergent-rinsing. The in vitro incubation conditions that most reflected the in vivo state required RNase inhibitor (purified from placenta), polyvinylpyrrolidone to prevent nuclear swelling, and addition of ATP. Under these circumstances, αAGP sequences were transported only from turpentine-stimulated preparations, were found only in poly(A) + RNA, and were the same size as authentic cytoplasmic mRNA. Omission of polyvinylpyrrolidone resulted in release of some αAGP sequences in smaller, more heterogeneous poly(A)-RNA, and leakage of some αAGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature αAGP mRNA to the nucleus. In contrast to αAGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest αAGP intron was not found in RNA transported from treated nuclei in complete medium. The intron-containing fragments remained in the nucleus, largely in poly(A)- RNA of a size consistent with free intron. Some hybridization of intron sequences was observed with cytoplasmic and nuclear membrane-associated poly(A) + RNA preparations which may represent 3′-processing catabolites; leakage of these sequences was considerably greater in the absence of PVP. On the basis of densitometric estimates, a 5-fold increase in the amount of αAGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of αAGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of αAGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of αAGP in the acute phase response.

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