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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.

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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.

Neuroblastoma (NB) is a pediatric cancer characterized by remarkable cell heterogeneity within the tumor nodules. Here, we demonstrate that the synthesis of a pol III-transcribed noncoding (nc) RNA (NDM29) strongly restricts NB development by promoting cell differentiation, a drop of malignancy processes, and a dramatic reduction of the tumor initiating cell (TIC) fraction in the NB cell population. Notably, the overexpression of NDM29 also confers to malignant NB cells an unpredicted susceptibility to the effects of antiblastic drugs used in NB therapy. Altogether, these results suggest the induction of NDM29 expression as possible treatment to increase cancer cells vulnerability to therapeutics and the measure of its synthesis in NB explants as prognostic factor of this cancer type.-Castelnuovo, M., Massone, S., Tasso, R., Fiorino, G., Gatti, M., Robello, M., Gatta, E., Berger, A., Strub, K., Florio, T., Dieci, G., Cancedda, R., Pagano, A. An Alu-like RNA promotes cell differentiation and reduces malignancy of human neuroblastoma cells. FASEB J. 24, 000 -000 (2010). www.fasebj.org