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

The transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidaz ole (DRB) is an adenosine analog that has been shown to cause premature tra nscriptional termination and thus has been a useful tool to identify factor s 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: in tron stabilization and the induction of long transcripts by a mechanism oth er than premature termination. We found that DRB had a biphasic effect on T -cell receptor-beta (TCR beta) transcripts driven by a tetracycline (tet)-r esponsive promoter in transfected HeLa cells. In the first phase, DRB cause d a rapid decrease (within five minutes) of pre-mRNA and its spliced intron (IVS1(C beta 1)), consistent with the known ability of DRB to inhibit tran scription. In the second phase (which began ten minutes to two hours after treatment, depending on the dose), DRB dramatically increased the levels of IVS1(C beta 1)-containing transcripts by a mechanism requiring de novo RNA synthesis. DRB induced the appearance of short 0.4 to 0.8 kb TCR beta tran scripts in vivo, indicating DRB enhances premature transcriptional terminat ion. A similar to 475 nt prematurely terminated transcript (PT) was charact erized that terminated at an internal poly(A) tract in the intron IVS1(C be ta 1). We identified three other effects of DRB. First, we observed that DR B induced the appearance of heterodisperse TCR beta transcripts that were t oo long (similar to 1 kb to >8 kb) to result from the type of premature ter mination events previously described. Their production was not promoter-spe cific, as we found that long transcripts were induced by DRB from both the tet-responsive and beta-actin promoters. Second, DRB upregulated full-lengt h normal-sized c-myc mRNA, which provided further evidence that DRB has eff ects besides regulation of premature termination. Third, DRB stabilized lar iat forms of the intron IVS1(C beta 1), indicating that DRB exerts post-tra nscriptional actions. We propose that our model system will be useful for e lucidating the factors that regulate RNA decay and transcriptional elongati on in vivo. (C) 2000 Academic Press.