ATP is dispensable for both miRNA- and Smaug-mediated deadenylation reactions.

MicroRNAs (miRNAs), as well as the RNA-binding protein Smaug, recruit the CCR4-NOT deadenylase complex for shortening of the poly(A) tail. It has been believed that ATP is required for deadenylation induced by miRNAs or Smaug, based on the fact that the deadenylation reaction is blocked by ATP depletion. However, when isolated, neither of the two deadenylases in the CCR4-NOT complex requires ATP by itself. Thus, it remains unknown why ATP is required for deadenylation by ribonucleoprotein complexes like miRNAs and Smaug. Herein we found that, in the absence of the ATP-regenerating system, ATP is rapidly consumed into AMP, a strong deadenylase inhibitor, in Drosophila cell lysate. Importantly, hydrolysis of AMP was sufficient to reactivate deadenylation by miRNAs or Smaug, suggesting that AMP accumulation, rather than ATP depletion, caused the inhibition of the deadenylation reaction. Our results indicate that ATP is dispensable for deadenylation induced by miRNAs or Smaug and emphasize caution in the use of ATP depletion methods.

CCR4 and CAF1 deadenylases have an intrinsic activity to remove the post-poly(A) sequence.

MicroRNAs (miRNAs) recruit the CCR4-NOT complex, which contains two deadenylases, CCR4 and CAF1, to promote shortening of the poly(A) tail. Although both CCR4 and CAF1 generally have a strong preference for poly(A) RNA substrates, it has been reported from yeast to humans that they can also remove non-A residues in vitro to various degrees. However, it remains unknown how CCR4 and CAF1 remove non-A sequences. Herein we show that Drosophila miRNAs can promote the removal of 3'-terminal non-A residues in an exonucleolytic manner, but only if an upstream poly(A) sequence exists. This non-A removing reaction is directly catalyzed by both CCR4 and CAF1 and depends on the balance between the length of the internal poly(A) sequence and that of the downstream non-A sequence. These results suggest that the CCR4-NOT complex has an intrinsic activity to remove the 3'-terminal non-A modifications downstream from the poly(A) tail.

RNA processing factors Swd2.2 and Sen1 antagonize RNA Pol III-dependent transcription and the localization of condensin at Pol III genes.

Condensin-mediated chromosome condensation is essential for genome stability upon cell division. Genetic studies have indicated that the association of condensin with chromatin is intimately linked to gene transcription, but what transcription-associated feature(s) direct(s) the accumulation of condensin remains unclear. Here we show in fission yeast that condensin becomes strikingly enriched at RNA Pol III-transcribed genes when Swd2.2 and Sen1, two factors involved in the transcription process, are simultaneously deleted. Sen1 is an ATP-dependent helicase whose orthologue in Saccharomyces cerevisiae contributes both to terminate transcription of some RNA Pol II transcripts and to antagonize the formation of DNA:RNA hybrids in the genome. Using two independent mapping techniques, we show that DNA:RNA hybrids form in abundance at Pol III-transcribed genes in fission yeast but we demonstrate that they are unlikely to faciliate the recruitment of condensin. Instead, we show that Sen1 forms a stable and abundant complex with RNA Pol III and that Swd2.2 and Sen1 antagonize both the interaction of RNA Pol III with chromatin and RNA Pol III-dependent transcription. When Swd2.2 and Sen1 are lacking, the increased concentration of RNA Pol III and condensin at Pol III-transcribed genes is accompanied by the accumulation of topoisomerase I and II and by local nucleosome depletion, suggesting that Pol III-transcribed genes suffer topological stress. We provide evidence that this topological stress contributes to recruit and/or stabilize condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1. Our data challenge the idea that a processive RNA polymerase hinders the binding of condensin and suggest that transcription-associated topological stress could in some circumstances facilitate the association of condensin.