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

Genome-wide studies in S. cerevisiae reveal that the transcriptome includes numerous antisense RNAs as well as intergenic transcripts regulated by the exosome component Rrp6. We observed that upon the loss of Rrp6 function, two PHO84 antisense transcripts are stabilized, and PHO84 gene transcription is repressed. Interestingly, the same phenotype is observed in wild-type cells during chronological aging. Epistasis and chromatin immunoprecipitation experiments indicate that the loss of Rrp6 function is paralleled by the recruitment of Hda1 histone deacetylase to PHO84 and neighboring genes. However, histone deacetylation is restricted to PHO84, suggesting that Hda1 activity depends on antisense RNA. Accordingly, the knockdown of antisense production prevents PHO84 gene repression, even in the absence of Rrp6. Together, our data indicate that the stabilization of antisense transcripts results in PHO84 gene repression via a mechanism distinct from transcription interference and that the modulation of Rrp6 function contributes to gene regulation by inducing RNA-dependent epigenetic modifications.

Eukaryotic genomes are extensively transcribed, forming both messenger (m) and noncoding (nc) RNAs. ncRNAs made by RNA polymerase II (Pol II) often initiate from bidirectional promoters (nucleosome-depleted chromatin) that synthesise mRNA and ncRNA in opposite directions. We demonstrate that actively transcribed mRNA encoding genes by adopting a gene loop conformation, restrict divergent transcription of ncRNAs. Since gene loop formation depends on a protein factor (Ssu72) that co-associates with both promoter and terminator, its inactivation leads to increased synthesis of promoter-associated divergent ncRNAs, referred to as Ssu72 restricted transcripts (SRT). Similarly, inactivation of individual gene loops by gene mutation enhances SRT synthesis. We demonstrate that gene loop conformation enforces transcriptional directionality on otherwise bidirectional promoters.

The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appearance in the cytoplasm, without restoring bulk poly(A)+ RNA export. Chromatin immunoprecipitation, FISH and pulse-chase experiments indicate that Mlps downregulate LacZ mRNA synthesis in a yra1 mutant strain. Microarray analyses reveal that Mlp2p also reduces a subset of cellular transcripts in the yra1 mutant. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlps rescues the growth defect of yra1 and nab2 but not other mRNA export mutants. We propose that Nab2p and Yra1p are required for proper mRNP docking to the Mlp platform. Defects in Yra1p prevent mRNPs from crossing the Mlp gate and this block negatively feeds back on the transcription of a subset of genes, suggesting that Mlps link mRNA transcription and export.

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.