Genome-wide pervasive transcription has been reported in many eukaryotic organisms, revealing a highly interleaved transcriptome organization that involves hundreds of previously unknown non-coding RNAs. These recently identified transcripts either exist stably in cells (stable unannotated transcripts, SUTs) or are rapidly degraded by the RNA surveillance pathway (cryptic unstable transcripts, CUTs). One characteristic of pervasive transcription is the extensive overlap of SUTs and CUTs with previously annotated features, which prompts questions regarding how these transcripts are generated, and whether they exert function. Single-gene studies have shown that transcription of SUTs and CUTs can be functional, through mechanisms involving the generated RNAs or their generation itself. So far, a complete transcriptome architecture including SUTs and CUTs has not been described in any organism. Knowledge about the position and genome-wide arrangement of these transcripts will be instrumental in understanding their function. Here we provide a comprehensive analysis of these transcripts in the context of multiple conditions, a mutant of the exosome machinery and different strain backgrounds of Saccharomyces cerevisiae. We show that both SUTs and CUTs display distinct patterns of distribution at specific locations. Most of the newly identified transcripts initiate from nucleosome-free regions (NFRs) associated with the promoters of other transcripts (mostly protein-coding genes), or from NFRs at the 3' ends of protein-coding genes. Likewise, about half of all coding transcripts initiate from NFRs associated with promoters of other transcripts. These data change our view of how a genome is transcribed, indicating that bidirectionality is an inherent feature of promoters. Such an arrangement of divergent and overlapping transcripts may provide a mechanism for local spreading of regulatory signals-that is, coupling the transcriptional regulation of neighbouring genes by means of transcriptional interference or histone modification

Many S. cerevisiae genes encode antisense transcripts some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single molecule resolution fluorescent in situ hybridization (smFISH) to investigate antisense-mediated transcription regulation. We show that PHO84 antisense RNA acts as a bimodal switch, where continuous low frequency antisense transcription represses sense expression within individual cells. Surprisingly, antisense RNAs do not accumulate at the PHO84 gene but are exported to the cytoplasm. Furthermore, loss of Rrp6, rather than stabilizing PHO84 antisense RNA, promotes antisense elongation by reducing its early transcription termination by Nrd1-Nab3-Sen1. These observations suggest that PHO84 silencing results from constant low frequency antisense transcription through the promoter rather than its static accumulation at the repressed gene.

Homology-dependent gene silencing, a phenomenon described as cosuppression in plants, depends on siRNAs. We provide evidence that in Saccharomyces cerevisiae, which is missing the RNAi machinery, protein coding gene cosuppression exists. Indeed, introduction of an additional copy of PHO84 on a plasmid or within the genome results in the cosilencing of both the transgene and the endogenous gene. This repression is transcriptional and position-independent and requires trans-acting antisense RNAs. Antisense RNAs induce transcriptional gene silencing both in cis and in trans, and the two pathways differ by the implication of the Hda1/2/3 complex. We also show that trans-silencing is influenced by the Set1 histone methyltransferase, which promotes antisense RNA production. Finally we show that although antisense-mediated cis-silencing occurs in other genes, transsilencing so far depends on features specific to PHO84. All together our data highlight the importance of noncoding RNAs in mediating RNAi-independent transcriptional gene silencing.[Keywords: Antisense RNA; cis and trans transcriptional gene silencing; PHO84; cosuppression; RNAi-independent TGS; noncoding RNA; S. cerevisiae] Supplemental material is available at http://www.genesdev.org.

Most genomes, including yeast Saccharomyces cerevisiae, are pervasively transcribed producing numerous non-coding RNAs, many of which are unstable and eliminated by nuclear or cytoplasmic surveillance pathways. We previously showed that accumulation of PHO84 antisense RNA (asRNA), in cells lacking the nuclear exosome component Rrp6, is paralleled by repression of sense transcription in a process dependent on the Hda1 histone deacetylase (HDAC) and the H3K4 histone methyl transferase Set1. Here we investigate this process genome-wide and measure the whole transcriptome of various histone modification mutants in a Δrrp6 strain using tiling arrays. We confirm widespread occurrence of potentially antisense-dependent gene regulation and identify three functionally distinct classes of genes that accumulate asRNAs in the absence of Rrp6. These classes differ in whether the genes are silenced by the asRNA and whether the silencing is HDACs and histone methyl transferase-dependent. Among the distinguishing features of asRNAs with regulatory potential, we identify weak early termination by Nrd1/Nab3/Sen1, extension of the asRNA into the open reading frame promoter and dependence of the silencing capacity on Set1 and the HDACs Hda1 and Rpd3 particularly at promoters undergoing extensive chromatin remodelling. Finally, depending on the efficiency of Nrd1/Nab3/Sen1 early termination, asRNA levels are modulated and their capability of silencing is changed.