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.

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Nucleolar channel systems (NCSs) are membranous organelles appearing transiently in the epithelial cell nuclei of postovulatory human endometrium. Their characterization and use as markers for a healthy receptive endometrium have been limited because they are only identifiable by electron microscopy. Here we describe the light microscopic detection of NCSs using immunofluorescence. Specifically, the monoclonal nuclear pore complex antibody 414 shows that NCSs are present in about half of all human endometrial epithelial cells but not in any other cell type, tissue or species. Most nuclei contain only a single NCS of uniform 1 μm diameter indicating a tightly controlled organelle. The composition of NCSs is as unique as their structure; they contain only a subset each of the proteins of nuclear pore complexes, inner nuclear membrane, nuclear lamina and endoplasmic reticulum. Validation of our robust NCS detection method on 95 endometrial biopsies defines a 6-day window, days 19-24 (±1) of an idealized 28 day cycle, wherein NCSs occur. Therefore, NCSs precede and overlap with the implantation window and serve as potential markers of uterine receptivity. The immunodetection assay, combined with the hitherto underappreciated prevalence of NCSs, now enables simple screening and further molecular and functional dissection.

RNA polymerase (pol) III, assisted by the transcription factors TFIIIC and TFIIIB, transcribes small untranslated RNAs, such as tRNAs. In addition to known pol III-transcribed genes, the Saccharomyces cerevisiae genome contains loci (ZOD1, ETC1-8) associated to incomplete pol III transcription complexes (Moqtaderi, Z., and Struhl, K. (2004) Mol. Cell. Biol. 24, 4118 -4127). We show that a short segment of the ZOD1 locus, containing box A and box B promoter elements and a termination signal between them, directs the pol III-dependent production of a small RNA both in vitro and in vivo. In yeast cells, the levels of both ZOD1-and ETC5-specific transcripts were dramatically enhanced upon nucleosome depletion. Remarkably, transcription factor and pol III occupancy at the corresponding loci did not change significantly upon derepression, thus suggesting that chromatin opening activates poised pol III to transcription. Comparative genomic analysis revealed that the ZOD1 promoter is the only surviving portion of a tDNA Ile ancestor, whose transcription capacity has been preserved throughout evolution independently from the encoded RNA product. Similarly, another TFIIIC/TFIIIB-associated locus, close to the YGR033c open reading frame, was found to be the strictly conserved remnant of an ancient tDNA Arg . The maintenance, by eukaryotic genomes, of chromatin-repressed, non-coding transcription units has implications for both genome expression and organization.In eukaryotes, RNA polymerase (pol) 3 III, assisted by a specific set of basal transcription factors (TFIIIA, TFIIIB, TFIIIC), transcribes at high efficiency the genes for tRNAs, 5 S rRNA, and a few other non-translated RNAs. In Saccharomyces cerevisiae, these include the RNA component of RNase P, the U6 small nuclear RNA, and the cytoplasmic RNA of the signal recognition particle, encoded respectively by the RPR1, SNR6, and SCR1 genes (1-3), as well as a small RNA of unknown function encoded by the RNA170 locus (4). The genomes of higher eukaryotes also contain a variety of extremely abundant, repetitive short interspersed elements (SINEs) that have evolved from tRNA or 7SL RNA genes and maintain a pol III promoter (5). SINE transcription by pol III can influence the expression of other genes, both at the transcriptional and at the post-transcriptional levels (5, 6). Up to now, no tRNA gene-derived transcription unit has been identified in yeast genomes. Genomewide chromatin immunoprecipitation analyses in S. cerevisiae have recently revealed several new loci that are associated to the pol III transcription machinery (7-9). One of them, SNR52, encodes a C/D box small nucleolar RNA. Other newly identified loci appear to be associated to incomplete transcription machinery. One of these loci has been named ZOD1 (for zone of disparity) because its occupancy by pol III was found to be disproportionately low when compared with TFIIIC occupancy (9). In another study, the same locus (referred to as iYML089c) was found to be associated to appreciable levels of pol III (8). ...

The Saccharomyces cerevisiae SNR52 gene is unique among the snoRNA coding genes in being transcribed by RNA polymerase III. The primary transcript of SNR52 is a 250-nucleotide precursor RNA from which a long leader sequence is cleaved to generate the mature snR52 RNA. We found that the box A and box B sequence elements in the leader region are both required for the in vivo accumulation of the snoRNA. As expected box B, but not box A, was absolutely required for stable TFIIIC, yet in vitro. Surprisingly, however, the box B was found to be largely dispensable for in vitro transcription of SNR52, whereas the box A-mutated template effectively recruited TFIIIB; yet it was transcriptionally inactive. Even in the complete absence of box B and both upstream TATA-like and T-rich elements, the box A still directed efficient, TFIIIC-dependent transcription. Box B-independent transcription was also observed for two members of the tRNA Asn (GTT) gene family, but not for two tRNA Pro (AGG) gene copies. Fully recombinant TFIIIC supported box B-independent transcription of both SNR52 and tRNA Asn genes, but only in the presence of TFIIIB reconstituted with a crude B؆ fraction. Non-TFIIIB component(s) in this fraction were also required for transcription of wild-type SNR52. Transcription of the box B-less tRNA Asn genes was strongly influenced by their 5-flanking regions, and it was stimulated by TBP and Brf1 proteins synergistically. The box A can thus be viewed as a core TFIIICinteracting element that, assisted by upstream TFIIIB-DNA contacts, is sufficient to promote class III gene transcription.RNA polymerase (Pol) 3 III synthesizes tRNA, 5 S rRNA, and a variety of other types of small nuclear and cytoplasmic RNAs. In general, the transcription of class III genes is under the control of internal control regions (ICR) characterized by discontinuous structures, with essential boxes separated by non-essential nucleotides (1). In the case of tRNA and 5 S rRNA genes, the ICRs are highly conserved and comprise the binding sites for the general transcription factor TFIIIC (box A and box B) and for the 5 S-specific factor TFIIIA (box C). Once assembled, TFIIIC recruits TFIIIB upstream of the transcription start site (TSS); TFIIIB in turn recruits Pol III for transcription initiation. The strong conservation of the ICRs likely reflects their dual function as both nucleation sites for transcription complex assembly and key determinants of tRNA and 5 S rRNA structure. An indication of the constraints imposed on ICR by their overlapping structural and functional roles comes from the variability of promoter organizations displayed by the minority of class III genes not coding for tRNA and 5 S rRNA. In some of these genes, the TFIIIC-interacting control regions (box A and box B) have been maintained within the transcribed region, and adapted to the structure of the small RNA without losing the transcriptional function. For example, in the Saccharomyces cerevisiae SCR1 gene, coding for the 7SL RNA, box A and box B are both intragenic an...