Dichlorobenzimidazole‐riboside Inhibition of Nuclear RNA Accumulation Initiated with γ‐Thio Analogues of ATP and GTP

Abstract

The y-thio analogues of ATP and GTP, ATP [S] and GTP [S], have been used as affinity probes to measure RNA synthesis initiated in vifro in L cell nuclei. 5,6-Dichlororibofuranosylbenzimidazole was found to inhibit total RNA synthesis in vitro and the amount bound by mercury-affinity chromatography. 5,6-Dichloro-l-/L~-ribofuranosylbenzimidazole has been shown to inhibit transcription of eucaryotic heterogeneous nuclear RNA (hnRNA) [l -31 in several cell lines, resulting in almost complete cessation of mRNA appearance in the cytoplasm [3]. Although evidence has been presented that the mechanism of this inhibition of RNA synthesis is by a process of premature termination in late adenovirus transcription 141 and HeLa cells [5], it has been difficult to study its effects on transcription in detail.The L cell nuclear system has been shown to be sensitive to 5,6-dichlororibofuranosylbenzimidazole inhibition of transcription in vitro [6] and might be expected to provide a convenient system in which to investigate the effect on RNA initiated in vitro. The y-thio analogues of purine triphosphates, ATP [S] and GTP [S] have been used previously to measure initiation of RNA in isolated mouse myeloma nuclei [7], Xenopus luevis oocyte nuclei [8] and adenovirus-infected cell nuclei [9]. Resistance of the [y-thioltriphosphates to phosphatases [7,10] allows isolation of the triphosphorylated termini from RNA molecules initiated in vitro by mercury affinity chromatography and even permits characterization of some of the products initiated in vitro [7-91. This paper reports the use of ATP[S] and GTP[S] to detect initiation in mouse L cell nuclei by means of Hg-affinity chromatography and direct incorporation of 3sS-labeled purine 5'-[1;-thio]triphosphate. Accumulation of RNA transcripts initiated in vitro was significantly inhibited by 5,6-dichlororibofuranosylbenzimidazole as measured by both of these methods. MATERIALS AND METHODS Isolation qf Nuclear R N A Labeled in vitroMouse L cell nuclei were prepared as described previously, after pretreatment of cells in vivo with low levels of actino- Boehringer Mannheim supplied the unlabeled [;!-thioltriphosphates; 5,6-dichlororibofuranosylhenzimidazole was a gift from D r A. F. Wagner (Merck, Sharp and Dohme) and was used at 350 pM. This inhibitor concentration was found to give more consistent results between different nuclear preparations used at 1 . 5~ 10' nuclei ml than the 100 pM concentrations used in previous experiments [6].R N A was extracted and separated from mononucleotides on a Sephadex G-50 column as reported [ I l l with the entire polynucleotide peak precipitated by 2.5 vol. ethanol and used in subsequent analyses. RNA Fractionat ionRNA was separated according to s i x class, after extensive denaturation in 80 dimethylsulfoxide ( Me2SO) for 2 min at 60 "C, by sucrose/sodium dodecyl sulfate gradient centrifugation [ll].Mercury affinity chromatography was carried out by adsorbing extracted RNA on an Affigel 50 1 (Bio-Rad) column in the presence of 1 mM EDTA as descr...