In vitro release of alpha 1-acid glycoprotein RNA sequences shows fidelity with the acute phase response in vivo.
The acute phase reaction of rat liver to subcutaneous turpentine challenge results in a 20- to 100-fold increase in alpha 1-acid glycoprotein (alpha AGP) mRNA. We utilized this response to establish conditions appropriate for study of RNA transport in vitro using hybridization with 32P-labeled exon and intron alpha AGP 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, alpha AGP 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 alpha AGP sequences in smaller, more heterogeneous poly(A)- RNA, and leakage of some alpha AGP sequences was observed from control preparations. Omission of ATP resulted in restriction of mature alpha AGP mRNA to the nucleus. In contrast to alpha AGP mRNA, transport of albumin mRNA was decreased 3-4X in turpentine-treated preparations. The largest alpha AGP 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 alpha AGP exon sequences was observed in nuclear RNA, comparing treated with control animals, but transport of alpha AGP exon sequences was detectable only from treated nuclei, indicating at least a 50-fold increase in abundance of alpha AGP sequences. This suggests that a selective gating mechanism may be operative at the level of post-transcriptional nucleocytoplasmic transport during induction of alpha AGP in the acute phase response.
D000818:Animals; D001483:Base Sequence; D005091:Exons; D005796:Genes / Q000187:drug effects; D007438:Introns; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D009961:Orosomucoid / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification; D051381:Rats; D014425:Turpentine / Q000494:pharmacology
D009961:Orosomucoid; D012333:RNA, Messenger; D014425:Turpentine