Rapid induction of nuclear transcripts and inhibition of intron decay in response to the polymerase II inhibitor DRB

Abstract

The transcriptional inhibitor 5,6-dichloro-1-β- d-ribofuranosylbenzimidazole (DRB) is an adenosine analog that has been shown to cause premature transcriptional termination and thus has been a useful tool to identify factors important for transcriptional elongation. Here, we establish an efficient system for studying DRB-sensitive steps of transcriptional elongation. In addition, we establish two novel effects of DRB not previously reported: intron stabilization and the induction of long transcripts by a mechanism other than premature termination. We found that DRB had a biphasic effect on T-cell receptor-β (TCRβ) transcripts driven by a tetracycline (tet)-responsive promoter in transfected HeLa cells. In the first phase, DRB caused a rapid decrease (within five minutes) of pre-mRNA and its spliced intron (IVS1 Cβ1), consistent with the known ability of DRB to inhibit transcription. In the second phase (which began ten minutes to two hours after treatment, depending on the dose), DRB dramatically increased the levels of IVS1 Cβ1-containing transcripts by a mechanism requiring de novo RNA synthesis. DRB induced the appearance of short 0.4 to 0.8 kb TCRβ transcripts in vivo, indicating DRB enhances premature transcriptional termination. A ∼475 nt prematurely terminated transcript (PT) was characterized that terminated at an internal poly(A) tract in the intron IVS1 Cβ1. We identified three other effects of DRB. First, we observed that DRB induced the appearance of heterodisperse TCRβ transcripts that were too long (∼1 kb to >8 kb) to result from the type of premature termination events previously described. Their production was not promoter-specific, as we found that long transcripts were induced by DRB from both the tet-responsive and β-actin promoters. Second, DRB upregulated full-length normal-sized c- myc mRNA, which provided further evidence that DRB has effects besides regulation of premature termination. Third, DRB stabilized lariat forms of the intron IVS1 Cβ1, indicating that DRB exerts post-transcriptional actions. We propose that our model system will be useful for elucidating the factors that regulate RNA decay and transcriptional elongation in vivo.