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N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

Intrinsic signals and cues from the external environment drive cell fate decisions. In budding yeast, the decision to enter meiosis is controlled by nutrient and mating-type signals that regulate expression of the master transcription factor for meiotic entry, IME1. How nutrient signals control IME1 expression remains poorly understood. Here we show that IME1 transcription is regulated by multiple sequence-specific transcription factors that mediate association of Tup1-Cyc8 co-repressor to its promoter. We find that at least eight transcription factors bind the IME1 promoter when nutrients are ample. Remarkably, association of these transcription factors is highly regulated by different nutrient cues. Mutant cells lacking three transcription factors (Sok2/Phd1/Yap6) displayed reduced Tup1-Cyc8 association, increased IME1 expression and earlier onset of meiosis. Our data demonstrate that the promoter of a master regulator is primed for rapid activation while repression by multiple transcription factors mediating Tup1-Cyc8 recruitment dictates the fate decision to enter meiosis.

Many long noncoding RNAs (lncRNAs) act in cis through transcription-coupled chromatin alterations that drive changes in local gene expression. How some cis-acting lncRNAs promote and others repress gene expression remains poorly understood. Here we report that in S. cerevisiae transcription levels of the lncRNA IRT2, located upstream in the promoter of the inducer of meiosis gene, regulate opposing chromatin and transcription states. Low IRT2 transcription displays enhancer RNA-like features. At these levels, IRT2 promotes histone exchange delivering acetylated histone H3 lysine 56 to chromatin thereby facilitating recruitment of a transcription factor and consequently activating transcription. Conversely, increasing IRT2 transcription enhances chromatin assembly and transcriptional repression. The opposing functions of IRT2 direct a regulatory circuit, which ensures that cells expressing opposite, but not one of either, mating-type loci enter meiosis. Our data demonstrate that the transcription levels of an lncRNA are key to controlling gene expression and cell fate outcomes.

SUMMARYLong Undecoded Transcript Isoforms (LUTIs) represent a class of non-canonical mRNAs that downregulate gene expression through the combined act of transcriptional and translational repression. While single gene studies revealed some important aspects of LUTI-based repression, how these features impact gene regulation at a global scale is unknown. By using transcript leader and direct RNA sequencing, here we identify 74 LUTI candidates that are expressed specifically during meiotic prophase. Translational repression of these candidates is ubiquitous and dependent on upstream open reading frames. However, LUTI-based transcriptional repression is highly variable. In only 50% of the cases, LUTI transcription causes downregulation of the protein-coding transcript isoform. Higher LUTI expression, enrichment of histone 3 lysine 36 trimethylation, and changes in nucleosome position are the strongest predictors of LUTI-based transcriptional repression. We conclude that LUTIs downregulate gene expression in a manner that integrates translational repression, chromatin state changes, and the magnitude of LUTI expression.

Promoter recognition by TATA-binding protein (TBP) is an essential step in the initiation of RNA polymerase II (pol II) mediated transcription. Genetic and biochemical studies in yeast have shown that Mot1p and NC2 play important roles in inhibiting TBP activity. To understand how TBP activity is regulated in a genome-wide manner, we profiled the binding of TBP, NC2, Mot1p, TFIID, SAGA, and pol II across the yeast genome using chromatin immunoprecipitation (ChIP)–chip for cells in exponential growth and during reprogramming of transcription. We find that TBP, NC2, and Mot1p colocalize at transcriptionally active pol II core promoters. Relative binding of NC2α and Mot1p is higher at TATA promoters, whereas NC2β has a preference for TATA-less promoters. In line with the ChIP–chip data, we isolated a stable TBP–NC2–Mot1p–DNA complex from chromatin extracts. ATP hydrolysis releases NC2 and DNA from the Mot1p–TBP complex. In vivo experiments indicate that promoter dissociation of TBP and NC2 is highly dynamic, which is dependent on Mot1p function. Based on these results, we propose that NC2 and Mot1p cooperate to dynamically restrict TBP activity on transcribed promoters.