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
SummarySen1 of S. cerevisiae is a known component of the NRD complex implicated in transcription termination of nonpolyadenylated as well as some polyadenylated RNA polymerase II transcripts. We now show that Sen1 helicase possesses a wider function by restricting the occurrence of RNA:DNA hybrids that may naturally form during transcription, when nascent RNA hybridizes to DNA prior to its packaging into RNA protein complexes. These hybrids displace the nontranscribed strand and create R loop structures. Loss of Sen1 results in transient R loop accumulation and so elicits transcription-associated recombination. SEN1 genetically interacts with DNA repair genes, suggesting that R loop resolution requires proteins involved in homologous recombination. Based on these findings, we propose that R loop formation is a frequent event during transcription and a key function of Sen1 is to prevent their accumulation and associated genome instability.
The function of non-coding antisense RNAs in yeast remains to be fully understood. Steinmetz and colleagues provide evidence for a general regulatory effect of antisense expression on sense genes and for a role in spreading regulatory signals between neighboring genes.
Several studies have shown that promoters of protein-coding genes are origins of pervasive non-coding RNA transcription and can initiate transcription in both directions. However, only recently have researchers begun to elucidate the functional implications of this bidirectionality and noncoding RNA production. Increasing evidence indicates that non-coding transcription at promoters influences the expression of protein-coding genes, revealing a new layer of transcriptional regulation. This regulation acts at multiple levels, from modifying local chromatin to enabling regional signal spreading and more distal regulation. Moreover, the bidirectional activity of a promoter is regulated at multiple points during transcription, giving rise to diverse types of transcripts.
Accurate estimates of mutation rates provide critical information to analyze genome evolution and organism fitness. We used whole-genome DNA sequencing, pulse-field gel electrophoresis, and comparative genome hybridization to determine mutation rates in diploid vegetative and meiotic mutation accumulation lines of Saccharomyces cerevisiae. The vegetative lines underwent only mitotic divisions while the meiotic lines underwent a meiotic cycle every ∼20 vegetative divisions. Similar base substitution rates were estimated for both lines. Given our experimental design, these measures indicated that the meiotic mutation rate is within the range of being equal to zero to being 55-fold higher than the vegetative rate. Mutations detected in vegetative lines were all heterozygous while those in meiotic lines were homozygous. A quantitative analysis of intra-tetrad mating events in the meiotic lines showed that inter-spore mating is primarily responsible for rapidly fixing mutations to homozygosity as well as for removing mutations. We did not observe 1–2 nt insertion/deletion (in-del) mutations in any of the sequenced lines and only one structural variant in a non-telomeric location was found. However, a large number of structural variations in subtelomeric sequences were seen in both vegetative and meiotic lines that did not affect viability. Our results indicate that the diploid yeast nuclear genome is remarkably stable during the vegetative and meiotic cell cycles and support the hypothesis that peripheral regions of chromosomes are more dynamic than gene-rich central sections where structural rearrangements could be deleterious. This work also provides an improved estimate for the mutational load carried by diploid organisms.
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