Degradation of Non-coding RNAs Promotes Recycling of Termination Factors at Sites of Transcription

Abstract

Highlights d Degradation of ncRNAs by the exosome frees bound termination factors Nab3 and Nrd1 d Exosome inhibition triggers accumulation of ncRNAs complexed with Nab3 and Nrd1 d Out-titration of Nab3 and Nrd1 by ncRNAs decreases NNS co-transcriptional binding d A circular RNA decoy recapitulates the global impairment of NNS-dependent termination

Data from crosslinking and analysis of cDNAs (CRAC) of Nab3 in yeast cells expressing a circular ncRNA decoy

“…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.…”

“…1 Schematic representation of the experimental rationale. As previously shown in the related research paper [1] , out-titration of the Nrd1-Nab3 RNA-binding module by excess ncRNA accumulating in exosome-impaired rrp6∆ cells or by expression of a circular ncRNA decoy leads to a decrease in its free pool (indicated by transparency). The overall CRAC signal is contributed by both co- and post-transcriptionally bound Nrd1-Nab3 to the RNA.…”

“…Yeast S.cerevisiae strain DLY3402 ( MATa NAB3::HTP::TRP1 dbr1∆::HIS3 ura3–1 ade2–1 leu2–3112 can1–100 ) carrying either the control pTet-i or the decoy pTet-i-CUT constructs (plasmids pDL865 and pDL866, respectively; [1] ) under control of the Tet OFF promoter were grown in CSM-URA-TRP medium supplemented with 2 μg/ml Doxycycline at 30 °C to mid-log phase, back diluted to OD 600 of 0.05 in 2 L CSM-URA-TRP medium without Doxycycline and grown to OD 600 of 0.6 at 30 °C before being subjected to UV crosslinking in vivo and subsequently processed for CRAC. Yeast S.pombe strain DLP01 ( h90 Rpb1::HTP::kan R MX ura4-DS/E ade6-M210 leu1–32 mat3M::ura4+ ) was grown in EMM+5AA medium to OD 600 of 0.5 at 30 °C before being subjected to UV crosslinking in vivo and subsequently processed for CRAC.…”

“…We have recently shown that the transcription termination control that the Nrd1-Nab3-Sen1 (NNS) complex exerts on pervasive nuclear non-coding RNAs (ncRNAs), ultimately leading to their degradation by the nuclear RNA exosome, can be impaired by the sequestration of termination factors by accumulation of ncRNAs [1] . We have shown that this build-up of ncRNAs out-titrating the Nrd1 and Nab3 NNS RNA-binding proteins and preventing their efficient recycling back to elongating RNA Polymerase II (RNAPII) could be induced either by inhibition of the exosome in yeast rrp6Δ cells or by expression of a circular ncRNA decoy ( Fig.…”

“…While in the related research paper we were able to show genomewide NNS-dependent termination impairment upon decoy expression through RNA-seq analysis, we were still missing direct proof that titration is occurring. Here, we present data from the analysis of nucleotide-resolution genomewide mapping of HTP-tagged Nab3 RNA binding in yeast cells expressing the circular ncRNA decoy designed to sequester Nab3 or a control construct [1] . Cells expressing the pTet-i-CUT and pTet-i constructs were pre-grown in the presence of doxycycline (i.e.…”