Nonsense-Mediated Decay Restricts LncRNA Levels in Yeast Unless Blocked by Double-Stranded RNA Structure

Abstract

SummaryAntisense long non-coding (aslnc)RNAs represent a substantial part of eukaryotic transcriptomes that are, in yeast, controlled by the Xrn1 exonuclease. Nonsense-Mediated Decay (NMD) destabilizes the Xrn1-sensitive aslncRNAs (XUT), but what determines their sensitivity remains unclear. We report that 3′ single-stranded (3′-ss) extension mediates XUTs degradation by NMD, assisted by the Mtr4 and Dbp2 helicases. Single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA mapping revealed that 3′-ss extensions discriminate the NMD-targeted XUTs from stable lncRNAs. Ribosome profiling showed that XUT are translated, locking them for NMD activity. Interestingly, mutants of the Mtr4 and Dbp2 helicases accumulated XUTs, suggesting that dsRNA unwinding is a critical step for degradation. Indeed, expression of anticomplementary transcripts protects cryptic intergenic lncRNAs from NMD. Our results indicate that aslncRNAs form dsRNA that are only translated and targeted to NMD if dissociated by Mtr4 and Dbp2. We propose that NMD buffers genome expression by discarding pervasive regulatory transcripts.

Ndt80 activates the meiotic ORC1 transcript isoform and SMA2 via a bi-directional middle sporulation element in Saccharomyces cerevisiae

“…In fact, this RNA turned out to be the Xrn1-sensitive unstable transcript XUT1538. 5 We note that the activity of Xrn1 is strong in S288C and W303 but attenuated in SK1 (Fig. 1B).…”

“…4 The mitotic isoform of ORC1 is divergently expressed with the long non-coding RNA XUT1538, which belongs to a class of regulatory lncRNAs that are targeted by the cytoplasmic 5 0 -3 0 exoribonuclease Xrn1. 5 Paradoxically, ORC1 expression is strongly induced in diploid cells that enter meiotic M-phase, 6,7 and this induction pattern coincides with the transcriptional activation of divergently expressed meiosis-specific SMA2. This gene is important for the spore membrane pathway that ensures proper encapsulation of haploid nuclei into spores.…”

“…S1 25 ) and the Saccharomyces Genomics Viewer (SGV, sgv.genouest.org 26 ). Furthermore, we interpreted DNA strand-specific RNA-Sequencing data from mitotically growing haploid and diploid wild type versus xrn1 mutant cells in S288C, W303 and SK1 strain backgrounds, 5 and our unpublished RNA-Sequencing data (not DNA strand-specific) from MATa/a and MATa/a cells cultured in YPD, YPA and SPII media (E. Becker, M. H. Guilleux, K. Waern, M. Snyder and M. Primig et al, in preparation).…”

“…The complete dataset was reported in reference. 5 (C) A heatmap like in panel A shows samples from diploid wild type cells cultured in rich media (YPD, YPA) and sporulation medium (SPII) taken at the time points indicated in hours (h). The strain is indicated to the right in green.…”

“…A DNA strand-specific RNA-Seq data set was used to interpret the transcriptomes of asynchronously growing haploid wild type and xrn1 temperature sensitive mutants in the S288C, W303, and SK1 backgrounds, and diploid SK1 wild type and xrn1 mutant cells. 5 …”