Specific small nuclear RNAs from SV40-transformed cells stimulate transcription initiation in nontransformed isolated nuclei

Abstract

Previous studies in our laboratory have implicated small nuclear RNAs (SnRNA) in the regulation of transcription in isolated mammalian cell nuclei. The present investigation was designed to develop a transcription assay system using isolated intact nuclei with optimized RNA polymerase II activity which would be capable of reinitiation in vitro to study the mode of action of the "active" RNA.We used nuclei isolated from either human WI38 or Balb 3T3 mouse cells to test the activity of SnRNA purified from SV40-transformed WI38 or 3T3 cell lines. These systems were found to support transcription up to 60 min, 40–60% of which was polymerase II dependent. In vitro initiations were detected by [γ-32P]ATP incorporation as well as by Hg-Sepharose chromatography using (γ-S)ATP as substrate. Results supported the following conclusions: (a) SnRNA from transformed cells stimulates the transcriptional activity of nontransformed nuclei while homologous SnRNA has little or no activity; (b) the stimulation is NaOH-sensitive and is dependent on RNA polymerase II since it is eliminated by 1 μg/mL α-amanitin; (c) the active subfraction of SnRNA from mouse cells was found to be of identical size (320–350 nucleotides) to that previously identified in human and monkey cells; and (d) analysis of the transcripts obtained from control and stimulated cell nuclei revealed that SnRNA activity is due primarily to an increase in the number of initiated chains.