Regulated antisense transcription controls expression of cell-type-specific genes in yeast.

Transcriptome profiling studies have recently uncovered a large number of noncoding RNA transcripts (ncRNAs) in eukaryotic organisms, and there is growing interest in their role in the cell. For example, in haploid Saccharomyces cerevisiae cells, the expression of an overlapping antisense ncRNA, referred to here as RME2 (Regulator of Meiosis 2), prevents IME4 expression. In diploid cells, the a1-α2 complex represses the transcription of RME2, allowing IME4 to be induced during meiosis. In this study we show that antisense transcription across the IME4 promoter region does not block transcription factors from binding and is not required for repression. Mutational analyses found that sequences within the IME4 open reading frame (ORF) are required for the repression mediated by RME2 transcription. These results support a model where transcription of RME2 blocks the elongation of the full-length IME4 transcript but not its initiation. We have found that another antisense transcript, called RME3, represses ZIP2 in a cell-type-specific manner. These results suggest that regulated antisense transcription may be a widespread mechanism for the control of gene expression and may account for the roles of some of the previously uncharacterized ncRNAs in yeast.

Arg-Pro-X-Ser/Thr is a consensus phosphoacceptor sequence for the meiosis-specific Ime2 protein kinase in Saccharomyces cerevisiae.

Ime2 is a meiosis-specific protein kinase in Saccharomyces cerevisiae that is functionally related to cyclin-dependent kinase. Although Ime2 regulates multiple steps in meiosis, only a few of its substrates have been identified. Here we show that Ime2 phosphorylates Sum1, a repressor of meiotic gene transcription, on Thr-306. Ime2 protein kinase assays with Sum1 mutants and synthetic peptides define a consensus Arg-Pro-X-Ser/Thr motif that is required for efficient phosphorylation by Ime2. The carboxyl residue adjacent to the phosphoacceptor (+1 position) also influences the efficiency of Ime2 phosphorylation with alanine being a preferred residue. This information has predictive value in identifying new potential Ime2 targets as shown by the ability of Ime2 to phosphorylate Sgs1 and Gip1 in vitro and could be important in differentiating mitotic and meiotic regulatory pathways.

Combined analysis of expression data and transcription factor binding sites in the yeast genome.

BACKGROUND: The analysis of gene expression using DNA microarrays provides genome wide profiles of the genes controlled by the presence or absence of a specific transcription factor. However, the question arises of whether a change in the level of transcription of a specific gene is caused by the transcription factor acting directly at the promoter of the gene or through regulation of other transcription factors working at the promoter. RESULTS: To address this problem we have devised a computational method that combines microarray expression and site preference data. We have tested this approach by identifying functional targets of the a1-alpha2 complex, which represses haploid-specific genes in the yeast Saccharomyces cerevisiae. Our analysis identified many known or suspected haploid-specific genes that are direct targets of the a1-alpha2 complex, as well as a number of previously uncharacterized targets. We were also able to identify a number of haploid-specific genes which do not appear to be direct targets of the a1-alpha2 complex, as well as a1-alpha2 target sites that do not repress transcription of nearby genes. Our method has a much lower false positive rate when compared to some of the conventional bioinformatic approaches. CONCLUSIONS: These findings show advantages of combining these two forms of data to investigate the mechanism of co-regulation of specific sets of genes.

Role of second-largest RNA polymerase I subunit Zn-binding domain in enzyme assembly.

The second-largest subunits of eukaryal RNA polymerases are similar to the beta subunits of prokaryal RNA polymerases throughout much of their lengths. The second-largest subunits from eukaryal RNA polymerases contain a four-cysteine Zn-binding domain at their C termini. The domain is also present in archaeal homologs but is absent from prokaryal homologs. Here, we investigated the role of the C-terminal Zn-binding domain of Rpa135, the second-largest subunit of yeast RNA polymerase I. Analysis of nonfunctional Rpa135 mutants indicated that the Zn-binding domain is required for recruitment of the largest subunit, Rpa190, into the RNA polymerase I complex. Curiously, the essential function of the Rpa135 Zn-binding domain is not related to Zn(2+) binding per se, since replacement of only one of the four cysteine residues with alanine led to the loss of Rpa135 function. Even more strikingly, replacement of all four cysteines with alanines resulted in functional Rpa135.

Interactions of the Mcm1 MADS box protein with cofactors that regulate mating in yeast.

The yeast Mcm1 protein is a member of the MADS box family of transcriptional regulatory factors, a class of DNA-binding proteins that control numerous cellular and developmental processes in yeast, Drosophila melanogaster, plants, and mammals. Although these proteins bind DNA on their own, they often combine with different cofactors to bind with increased affinity and specificity to their target sites. To understand how this class of proteins functions, we have made a series of alanine substitutions in the MADS box domain of Mcm1 and examined the effects of these mutations in combination with its cofactors that regulate mating in yeast. Our results indicate which residues of Mcm1 are essential for viability and transcriptional regulation with its cofactors in vivo. Most of the mutations in Mcm1 that are lethal affect DNA-binding affinity. Interestingly, the lethality of many of these mutations can be suppressed if the MCM1 gene is expressed from a high-copy-number plasmid. Although many of the alanine substitutions affect the ability of Mcm1 to activate transcription alone or in combination with the alpha 1 and Ste12 cofactors, most mutations have little or no effect on Mcm1-mediated repression in combination with the alpha 2 cofactor. Even nonconservative amino acid substitutions of residues in Mcm1 that directly contact alpha 2 do not significantly affect repression. These results suggest that within the same region of the Mcm1 MADS box domain, there are different requirements for interaction with alpha 2 than for interaction with either alpha1 or Ste12. Our results suggest how a small domain, the MADS box, interacts with multiple cofactors to achieve specificity in transcriptional regulation and how subtle differences in the sequences of different MADS box proteins can influence the interactions with specific cofactors while not affecting the interactions with common cofactors.