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CRAC of Nab3 in yeast cells accumulating a circular RNA containing Nab3 binding sites (the decoy) or an empty control

ABSTRACT: We report the analysis of in vivo, genomewide binding of Nab3 under conditions in which non-degradable targets of Nab3 accumulate. We aimed at mimicking the effect of defective degradation of non-coding RNAs (Cryptic Unstable Transcripts, CUT) that can be bound by Nab3 on the co-transcriptional recruitment of Nab3 for transcription termination.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE164587 | GEO | 2021/01/12

REPOSITORIES: GEO

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Project description:Pervasive transcription originating from the ubiquitous activity of RNA Polymerase II (RNAPII) generates a vast mass of non-coding RNAs (ncRNAs) that represent a potential harm to gene expression. In the compact genome of the yeast Saccharomyces cerevisiae, the main genomewide safeguard against pervasive ncRNAs is the Nrd1-Nab3-Sen1 (NNS) complex, composed of two RNA-binding proteins (Nrd1 and Nab3) and the helicase Sen1. The NNS complex directs transcription termination of ncRNA genes and promotes the rapid degradation of pervasive transcripts from yeast nuclei through its physical and functional coupling to the nuclear RNA exosome. We have recently shown that inhibition of the exosome in yeast cells leads to the accumulation of ncRNAs complexed with Nab3 and Nrd1, decreasing recycling of these termination factors to sites of transcription and inducing global termination defects at NNS targets. Consistent with the notion that ncRNAs out-titrate Nab3 and Nrd1 termination factors, we have shown that a similar genomewide termination impairment could be achieved by expressing a circular RNA decoy containing a Nab3 binding target [1]. In relation to this previous research article, here we expand our observations on the effect of the circular RNA decoy on NNS termination. We aimed at verifying that the Nab3 binding sequence present on the decoy is indeed efficiently sequestering Nab3 as intended by design, leading to the expected decrease of Nab3 binding on NNS targets. We employed the crosslinking and cDNA analysis protocol (CRAC) on yeast cells expressing the circular ncRNA decoy or a control construct. We present data from high-resolution genomewide RNA binding of Nab3 in three independent biological replicates of these S.cerevisiae cells, normalized by spiked-in S.pombe lysates. These data allow the useful assessment of the extent of co-transcriptional binding decrease of Nab3 by decoy ncRNA titration and will be valuable for further analyses of NNS targeting mechanisms.

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CRAC of Nab3 in yeast cells accumulating a circular RNA containing Nab3 binding sites (the decoy) or an empty control

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