Most known transcriptional regulators are proteins, and the mechanisms they use to control transcription are well characterized. In recent years, however, a number of RNAs that regulate transcription have been identified, which seem to use different mechanisms. Two reports in Nature Structural and Molecular Biology by the Kugel and Goodrich group describe the identification of one such RNA regulator — the mouse B2 RNA — which inhibits transcription by binding to RNA polymerase II (Pol II).

B2 is a small non-coding RNA that is transcribed by RNA polymerase III (Pol III). While studying the transcriptional response to heat shock — which is characterized by the overall repression of Pol-II-mediated transcription and the upregulation of some heat-shock genes such as Hsp70 — Kugel, Goodrich and co-workers found that the level of nuclear B2 RNA increases.

When they specifically inhibited Pol III, they found that the levels of B2 RNA and Pol-II-mediated transcription remained unchanged after heat shock. This indicates that Pol-III-mediated transcription is somehow involved in blocking Pol-II-mediated transcription. Indeed, B2 RNA inhibited Pol-II-mediated transcription in vitro, whereas in mouse cells that were depleted of B2 RNA, mRNA levels remained constant after heat shock.

Next, Kugel, Goodrich and colleagues set out to characterize the mechanism by which B2 RNA inhibits transcription. The addition of B2 RNA to a minimal reconstituted transcription system that contained purified Pol II and three general transcription factors caused a potent and specific inhibition of transcription. An increase in the concentration of Pol II in the system required the addition of a higher concentration of B2 RNA to inhibit transcription, whereas an excess of either of the general transcription factors did not affect the requirement for B2 RNA. Earlier, the authors had shown that B2 RNA co-immunoprecipitated with Pol II in a heat-shock-specific manner. So, together, these data confirm that B2 RNA targets Pol II directly to inhibit transcription. The binding site was found to be a previously characterized RNA-docking site on Pol II.

So, which step in the transcription process does B2 RNA block? To address this question, the authors added B2 RNA to the in vitro transcription reaction at different stages, and found that only when it was added before the formation of the transcription pre-initiation complex did B2 RNA block transcription completely. In addition, B2 RNA inhibited the production of very short (up to three-nucleotide) transcripts. So, Kugel, Goodrich and co-workers concluded that inhibition must occur at, or before, transcription initiation. They showed that, in mechanistic terms, this transcriptional inhibition involves the binding of B2 RNA to the pre-initiation complex at the promoter, which renders the complex non-functional.

Understanding how B2-RNA-mediated repression of transcription can be relieved is a challenge for the future — the authors have already shown that the effect of B2 RNA is reversible. Another interesting conundrum is how genes such as Hsp70 can be transcribed despite the presence of B2 RNA.