Antisense 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. Display omitted •Xrn1-sensitive Unstable Transcripts (XUTs) are 3′-extended isoforms of stable lncRNAs•Nonsense-Mediated Decay preferentially targets long XUTs with single-stranded 3′ end•Antisense XUTs form double-stranded RNA in vivo•Formation of double-stranded RNA protects XUTs from Nonsense-Mediated Decay Wery et al. used single-gene investigation, genome-wide RNA analyses, and double-stranded (ds)RNA in vivo mapping to show that antisense Xrn1-sensitive Unstable Transcripts (XUTs) form double-stranded RNA in yeast and that 3′ single-stranded extension mediates XUTs degradation by the Nonsense-Mediated Decay (NMD) pathway, assisted by the Mtr4 and Dbp2 RNA helicases.