Degradation of non-coding RNAs promotes recycling of termination factors at sites of transcription

Abstract

A large share of the non-coding transcriptome in yeast is controlled by the Nrd1-Nab3-Sen1 (NNS) complex, which promotes transcription termination of noncoding RNA (ncRNA) genes, and by the nuclear exosome, which limits the steady state levels of the transcripts produced. How global ncRNA levels impinge on cellular functions is a longstanding and important question. Here we show that degradation of ncRNAs by the exosome is required for freeing Nrd1 and Nab3 from the released transcript after termination. In exosome mutants, these factors are sequestered by ncRNAs and cannot be efficiently recycled to sites of transcription, which induces termination defects genome-wide. ncRNA-dependent, genome-wide termination defects can be recapitulated by the expression of a degradationresistant, circular RNA containing a natural NNS target in exosome proficient cells.Our results have important implications for the mechanism of termination, the general impact of ncRNAs on cellular physiology and the importance of nuclear ncRNA degradation.Government through its "Investments for the Future" program.This work has benefited from the facilities and expertise of the high throughput sequencing core facility of I2BC (Centre de Recherche de Gif -http://www.i2bc-saclay.fr/). AUTHORS CONTRIBUTION Conceptualization, D.L., T.V., and A.J.; Methodology, T.V., M.B., and M-J.M.N.; Software, M.B.; Formal Analysis, M.B., T.V., and D.L.; Investigation, T.V., M-J.M.N., and D.L.; Writing -Original Draft, T.V. and D.L.; Writing -Review and Editing, T.V. and D.L.; Funding Acquisition, D.L.; Supervision, D.L.

Altered rRNA processing disrupts nuclear RNA homeostasis via competition for the poly(A)-binding protein Nab2

“…In a similar way, it could be anticipated that a change in cellular state that causes alterations in mRNA or ncRNA processing would also feedback to impact genome-wide RNA maturation events through RBP availability. In agreement with this notion, recent work showed that stabilization of pervasive transcripts, such as CUTs in rrp6Δ cells, results in the sequestration of Nab3 and Nrd1 on these transcripts and titration of these RBPs away from other RNA substrates resulting in genome-wide transcription termination defects due to limited NNS availability ( 97 ). Similarly, it was proposed that the accumulation of Nab2 on mRNAs upon nuclear export failure prevents its recycling to newly generated RNA molecules, which leaves nascent transcripts vulnerable to nuclear decay ( 44 , 45 ).…”

The S. cerevisiae m6A-reader Pho92 impacts meiotic recombination by controlling key methylated transcripts

“…To identify m 6 A-dependent mRNA targets bound by Pho92 during meiotic recombination, we performed an improved version of the UV-Crosslinking and analysis of cDNAs (CRAC) technique (Candelli et al, 2018; Granneman et al, 2009; Villa et al, 2020) on samples harvested at 5 hr in SPO medium. As required for the procedure, we used cells expressing an HTP (His 6 -TEV-ProteinA)-tagged version of Pho92 in WT and in ime4Δ / Δ strain as a control, to assess its specific binding to m 6 A-modified RNAs.…”

“…CRAC was performed essentially as described (Villa et al, 2020) with minor modifications. To facilitate later normalization between samples during dataset analysis, we used an internal “spike-in” extract of S. pombe expressing an RNA-binding protein fused to the HTP tag.…”

Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure

“…In rrp6Δ and other RNA exosome deletion mutant backgrounds, AS transcription is constitutively induced due to sequestration of the NNS (Nrd1-Nab3-Sen1) termination complex by stabilised non-coding RNAs. The NNS complex cannot be efficiently recycled to sites of transcription, inducing termination defects at non-coding RNA loci and resulting in their increased elongation frequency (48). To rule out possible indirect effects on transcription of the PHO5 gene due to gene deletion mutant backgrounds in which AS transcription is constitutively elongated, we turned to a system in which AS elongation is inducible.…”

“…To this end, we used the Anchor Away (AA) system to rapidly deplete Nrd1 protein from the nucleus by rapamycin treatment (50). Since Nrd1 belongs to the NNS surveillance system, its removal is expected to trigger transcriptional read-through of non-coding RNAs (49). Indeed, treatment of Nrd1-AA cells with rapamycin resulted in rapid induction of the PHO5 AS transcript production, clearly demonstrating that the NNS complex is important for its early termination in wild-type cells.…”