More strangeness …

Awhile ago, I discussed a flurry of papers in Science that showed some curious aspects of transcription and promoters.  It seems as if every passing day brings a new report that pertains to the phenomenon.  A recent issue of Nature brings us two papers, back to back, that are relevant.  The bottom line is that bidirectional transcription is a widespread phenomenon, at least in yeast.  Moreover, this phenomenon is responsible, not just for divergent transcription of mRNA-encoding genes, but also for the production of so-called Cryptic Unstable Transcripts and other uncharacterized RNAs.  The abstracts and some brief commentary are beneath the fold.

From a paper by Xu et al:

Genome-wide pervasive transcription has been reported in many eukaryotic organisms (1–7), revealing a highly interleaved transcriptome organization that involves hundreds of previously unknown non-codingRNAs8. 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 function9. Single-gene studies have shown that transcription of SUTs and CUTs can be functional, through mechanisms involving the generated RNAs10,11 or their generation itself12–14. 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 function8,15. 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 39 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.

The following report, by Neil et al:

Pervasive and hidden transcription is widespread in eukaryotes (1–4), but its global level, the mechanisms from which it originates and its functional significance are unclear. Cryptic unstable transcripts (CUTs) were recently described as a principal class of RNA polymerase II transcripts in Saccharomyces cerevisiae (5). These transcripts are targeted for degradation immediately after synthesis by the action of the Nrd1–exosome–TRAMP complexes (6,7). Although CUT degradation mechanisms have been analysed in detail, the genome-wide distribution at the nucleotide resolution and the prevalence of CUTs are unknown. Here we report the first high-resolution genomic map of CUTs in yeast, revealing a class of potentially functional CUTs and the intrinsic bidirectional nature of eukaryotic promoters. An RNA fraction highly enriched in CUTs was analysed by a 39 Long-SAGE (serial analysis of gene expression) approach adapted to deep sequencing. The resulting detailed genomic map of CUTs revealed that they derive from extremely widespread and very well defined transcription units and do not result from unspecific transcriptional noise. Moreover, the transcription of CUTs predominantly arises within nucleosome-free regions, most of which correspond to promoter regions of bona fide genes. Some of the CUTs start upstream from messenger RNAs and overlap their 59 end. Our study of glycolysis genes, as well as recent results from the literature8–11, indicate that such concurrent transcription is potentially associated with regulatory mechanisms. Our data reveal numerous new CUTs with such a potential regulatory role. However, most of the identified CUTs corresponded to transcripts divergent from the promoter regions of genes, indicating that they represent by-products of divergent transcription occurring at many and possibly most promoters. Eukaryotic promoter regions are thus intrinsically bidirectional, a fundamental property that escaped previous analyses because in most cases divergent transcription generates short-lived unstable transcripts present at very low steady-state levels.

What these two reports describe is a very high frequency of divergent transcription, originating from genomic positions that are depleted for nucleosomes.  Xu et al. conducted a large-scale meta-analysis of tiling array studies done in yeast (Saccharomyces cerevisiae) along with other reports of the distribution of nucleosomes in the yeast genome; so-called cryptic unstable transcripts (or CUTs) were identified by their increased abundance in mutants deficient in an exosome subunit (RRP6).   (See this essay for an introduction of sorts to the concept of CUTs and the interplay with the exosome.)  Neil, in contrast, purified CUTs by taking advantage of their increased abundance in yeast strains deficient in two components of the nuclear complex that degrades these unstable RNAs (the nuclus-specific exosome subunit RRP6 and the degradation-associated poly(A) polymerase TRF4) and their association with the nuclear cap-binding complex; thus, they were able to affinity-purify a tagged form of the cap-binding complex from an rrp6/trf4 double mutant, convert the associated RNAs to cDNA, and analyze the population using a technique called “Serial Analysis of Gene Expression”, or SAGE.  (These authors used a variation called LongSAGE, so named because the pieces of DNA that are produced by the technique are longer than those produced by the “classical” SAGE method.)  These authors catalogued and analyzed these CUTs.

While the methods and interests of the two studies were somewhat different, they both reveal something very interesting – namely, that all manner of transcription seemingly can initiate at the nucleosome-poor regions that typify promoter regions.  This would include transcription that yields translatable mRNAs, transcription of unannotated but stable RNAs of unknown function, and transcription of so-called cryptic unstable RNAs.  This raises many interesting questions about the decisions that occur at promoters – why make a translatable mRNA instead of a CUT (or stable unannotated transcript, for that matter), is transcriptional regulation accompanied by changes in the balance of mRNA and CUT production, etc., etc.?

The references:

Zhenyu Xu, Wu Wei, Julien Gagneur, Fabiana Perocchi, Sandra Clauder-Munster, Jurgi Camblong, Elisa Guffanti, Francoise Stutz, Wolfgang Huber & Lars M. Steinmetz. 2009.
Bidirectional promoters generate pervasive transcription in yeast.  Nature 457, 1033.

Helen Neil, Christophe Malabat, Yves d’Aubenton-Carafa, Zhenyu Xu, Lars M. Steinmetz & Alain Jacquier. 2009. Widespread bidirectional promoters are the major source of cryptic transcripts in yeast.  Nature 457, 1038.

Postscript – I would point out that these two studies involve a somewhat different set of CUTs than those described in earlier studies (discussed here).  The older reports focused on regions of the yeast genome that were apparently devoid of annotated features, while these more recent studies intentionally look at annotated regions of the genome.  This is because the studies by Xu et al. and Neil et al. anchor their analyses on the regions immediately upstream and downstream from protein coding regions.  However, the studies by Xu et al. and Neil et al. provide one possible mechanism for the origination of CUTs in gene-free regions, namely that RNA polymerase II will, with a low but significant frequency, initiate transcription on DNA that is devoid of nucleosomes (for whatever reason).

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