Actinomycin D Stimulates the Transcription of rRNA Minigenes Transfected into Mouse Cells. Implications for the in vivo Hypersensitivity of rRNA Gene Transcription

Abstract

The in vivo hypersensitivity of eukaryotic rRNA gene transcription to actinomycin D has long beenknown, but this effect could not be reproduced in model systems and its molecular mechanisms remain uncertain. We studied the action of actinomycin D using mouse rRNA minigenes (with RNA polymerase I promoter and terminator signals), carrying truncated mouse or human rDNA inserts, which are faithfully transcribed upon transient transfection into mouse cells. Low concentrations (0.01–0.08 μg/ml) of actinomycin D caused within 1–2 h a 2–7-fold stimulation of the transcription of rRNA minigenes which is inversely related to the size of the rDNA transcript. With transcripts longer than 3 kb the effect was reversed and at 4 kb a practically complete inhibition of the formation of full-length transcripts was observed, accompanied, however, by an enhanced accumulation of unfinished rDNA transcripts. The dependence of actinomycin D action on transcript length was also observed with lacZ gene segments of different size inserted into the mouse rRNA minigenes. The transcription initiation of endogenous rRNA genes was also stimulated by the low doses of actinomycin D as indicated by the enhanced synthesis of unfinished rDNA transcripts (spanning mainly the 5′ external transcribed spacer), whereas the synthesis of full-length transcripts was abolished. Removal of actinomycin D from the medium caused within 8–24 h a dramatic increase of the transcription from all rRNA minigenes tested. This stimulation was also inversely related to the size of the transcripts and varied from twofold to fivefold for the 3–4-kb transcripts to about 50–80-fold for the basic minigene transcript (395 nucleotides). The amount of endogenous aborted rDNA transcripts was also markedly increased, but the synthesis of full-length transcripts was not restored even 24 h after removal of the drug. The present results reproduce in a model cellular system the in vivo hypersensitivity of rRNA gene transcription to actinomycin D and reveal that the major factor involved is the size of the rRNA gene transcript. This effect requires only the basic rRNA gene promoter and terminator signals and does not depend on the G+C content of the RNA polymerase I transcripts. We suggest that at low concentrations, the intercalation of actinomycin D changes the conformation of DNA in the promoter region in a manner that stimulates the transcription of both endogenous and transfected rRNA genes. However, transcriptiondriven positive supercoiling of rDNA is enhanced by actinomycin D and above a critical transcript length it causes a blockage of transcription elongation and the accumulation of aborted rDNA transcripts.