Cryptic Transcription Mediates Repression of Subtelomeric Metal Homeostasis Genes

Nonsense-mediated mRNA decay (NMD) prevents the accumulation of transcripts bearing premature termination codons. Here we show that Saccharomyces cerevisiae NMD mutants accumulate 5′–extended RNAs (CD-CUTs) of many subtelomeric genes. Using the subtelomeric ZRT1 and FIT3 genes activated in response to zinc and iron deficiency, respectively, we show that transcription of these CD-CUTs mediates repression at the bona fide promoters, by preventing premature binding of RNA polymerase II in conditions of metal repletion. Expression of the main ZRT1 CD-CUT is controlled by the histone deacetylase Rpd3p, showing that histone deacetylases can regulate expression of genes through modulation of the level of CD-CUTs. Analysis of binding of the transcriptional activator Zap1p and insertion of transcriptional terminators upstream from the Zap1p binding sites show that CD-CUT transcription or accumulation also interferes with binding of the transcriptional activator Zap1p. Consistent with this model, overexpressing Zap1p or using a constitutively active version of the Aft1p transcriptional activator rescues the induction defect of ZRT1 and FIT3 in NMD mutants. These results show that cryptic upstream sense transcription resulting in unstable transcripts degraded by NMD controls repression of a large number of genes located in subtelomeric regions, and in particular of many metal homeostasis genes

Identification of new RNA substrates for the NMD and exosome RNA surveillance pathways in Saccharomyces cerevisiae

Using the yeast, Saccharomyces cerevisiae, as a model system, we utilized Affymetrix tiling arrays to globally investigate novel targets of the nonsense mediated decay pathway. The research that has been presented in the first chapter of this dissertation is centered around the investigation of extended transcripts and the degradation pathways that contribute to its rapid turnover in yeast cells. This work was published in the online version of the journal, PLoS Genetics. The work that is presented in the second chapter is based on our findings that intron-retained or unspliced RNAs are markedly stabilized in NMD mutant strains which led us to conclude the widespread impact of the nonsense mediated decay pathway in the degradation of unspliced RNAs. We further expanded our work and showed that introns emanating from NMD-sensitive loci can elicit an NMD-sensitive phenotype in trans, which led us to conclude that the intron (intronic sequences) are necessary for proper NMD-targeting. We further discovered that sequences located within the 5'SS as well as the branchpoint (BP) are responsible in part for the generation of unspliced/inefficiently spliced transcripts. Lastly, we showed that amino acid starvation results in a transient inhibition of nuclear splicing resulting in hyperaccumulation of unspliced transcripts. Under this condition, we elucidated a stress-specific role for the NMD pathway in rapidly targeting and degrading these unspliced transcripts. The results that have been presented in the second chapter of this dissertation have been published in the Molecular Cell journal (August 8th, 2008 issue). The third chapter (part) of this dissertation explores themes that have directly evolved from the first part. Specifically, the second part has shown that there are more complex routes for the degradation for unspliced transcripts besides the NMD pathway. In particular, synergistic modes of degradation involving components of the nuclear exosome and factors involved in NMD function to limit and hence degrade these unspliced transcripts. Decay measurements were undertaken to show that there are unspliced RNAs whose degradation mode depends on a synergistic mode of degradation requiring both the nuclear exosome component, Rrp6p and the NMD factor, Upf1p. Conversely, we found a class of unspliced transcripts whose turnover relies largely on the action of the NMD pathway and not on synergistic degradation. We further expanded our research and showed that inactivation of an essential, conserved, yeast RNA export factor, Mex67p, results in the nuclear sequestration and degradation of normally cytoplasmically degraded unspliced transcripts. This result highlighted the generic features of RNA degradation and has shown that temporal changes (such as malfunction of proper nuclear-to-cytoplasmic RNA trafficking) cause spatial redistribution and degradation of the unspliced RNA. We have also shown that the nuclear periphery factor, Mlp1p, could have a potential role in the retention of nuclear-degraded unspliced RNA. Deletion of this factor led to an increase in the cytoplasmic concentration of the unspliced RNA which was then degraded by the NMD pathway