Stimulation of later functions of the yeast meiotic protein kinase Ime2p by the IDS2 gene product.

Ime2p is a protein kinase that is expressed only during meiosis in Saccharomyces cerevisiae. Ime2p stimulates early, middle, and late meiotic gene expression and down-regulates expression of IME1, which specifies an activator of early meiotic genes that acts independently of Ime2p. We have identified a new gene, IDS2 (for IME2-dependent signaling), which has a functional relationship to Ime2p. An ids2 null mutation delays down-regulation of IME1 and expression of middle and late meiotic genes. In an ime1 null mutant that express IME2 from the GAL1 promoter (ime1 delta PGAL1-IME2 mutant), early meiotic gene expression depends only upon Ime2p. In such strains, Ids2p is dispensable for expression of the early genes HOP1 and SPO13 but is essential for expression of the middle and late genes SPS1, SPS2, and SPS100. Ids2p is also essential for the autoregulatory pathway through which Ime2p activates its own expression via the IME2 upstream activation sequences (UAS). An PGAL1-IME2 derivative that produces a truncated Ime2p (lacking its C-terminal 174 residues) permits IME2 UAS activation in the absence of Ids2p. This observation suggests that Ids2p acts upstream of Ime2p or that Ids2p and Ime2p act in independent, convergent pathways to stimulate IME2 UAS activity. Accumulation of epitope-tagged Ids2p derivatives is greatest in growing cells and declines during meiosis. We propose that Ids2p acts indirectly to modify Ime2p activity, thus permitting Ime2p to carry out later meiotic functions.     

The Cdk-activating kinase Cak1p promotes meiotic S phase through Ime2p

CAK1 encodes an essential protein kinase in Saccharomyces cerevisiae that is required for activation of the Cdc28p Cdk. CAK1 also has several CDC28-independent functions that are unique to meiosis. The earliest of these functions is to induce S phase, which is regulated differently in meiosis than in mitosis. In mitosis, Cdc28p controls its own S-phase-promoting activity by signaling the destruction of its inhibitor, Sic1p. In meiosis, Sic1p destruction is signaled by the meiosis-specific Ime2p protein kinase. Our data show that Cak1p is required to activate Ime2p through a mechanism that requires threonine 242 and tyrosine 244 in Ime2p's activation loop. This activation promotes autophosphorylation and accumulation of multiply phosphorylated forms of Ime2p during meiotic development. Consistent with Cak1p's role in activating Ime2p, cells lacking Cak1p are deficient in degrading Sic1p. Deletion of SIC1 or overexpression of IME2 can partially suppress the S-phase defect in cak1 mutant cells, suggesting that Ime2p is a key target of Cak1p regulation. These data show that Cak1p is required for the destruction of Sic1p in meiosis, as in mitosis, but in meiosis, it functions through a sporulation-specific kinase.     

Positive control of sporulation-specific genes by the IME1 and IME2 products in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, meiosis and spore formation require the induction of sporulation-specific genes. Two genes are thought to activate the sporulation program: IME1 and IME2 (inducer of meiosis). Both genes are induced upon entry into meiosis, and IME1 is required for IME2 expression. We report here that IME1 is essential for expression of four sporulation-specific genes. In contrast, IME2 is not absolutely essential for expression of the sporulation-specific genes, but contributes to their rapid induction. Expression of IME2 from a heterologous promoter permits the expression of these sporulation-specific genes, meiotic recombination, and spore formation in the absence of IME1. We propose that the IME1 and IME2 products can each activate sporulation-specific genes independently. In addition, the IME1 product stimulates sporulation-specific gene expression indirectly through activation of IME2 expression.     

Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene

N⁶-Methyladenosine (m⁶A) is present at internal sites in mRNA isolated from all higher eukaryotes, but has not previously been detected in the mRNA of the yeast Saccharomyces cerevisiae. This nucleoside modification occurs only in a sequence- specific context that appears to be conserved across diverse species. The function of this modification is not fully established, but there is some indirect evidence that m⁶A may play a role in the efficiency of mRNA splicing, transport or translation. The S.cerevisiae gene IME4, which is important for induction of sporulation, is very similar to the human gene MT-A70, which has been shown to be a critical subunit of the human mRNA [N⁶-adenosine]-methyltransferase. This observation led to the hypothesis that yeast sporulation may be dependent upon methylation of yeast mRNA, mediated by Ime4p. In this study we show that induction of sporulation leads to the appearance of low levels of m⁶A in yeast mRNA and that this modification requires IME4. Moreover, single amino acid substitutions in the putative catalytic residues of Ime4p lead to severe sporulation defects in a strain whose sporulation ability is completely dependent on this protein. Collectively, these data suggest very strongly that the activation of sporulation by Ime4p is the result of its proposed methyltransferase activity and provide the most direct evidence to date of a physiologic role of m⁶A in a gene regulatory pathway.     

A transcriptional cascade governs entry into meiosis in Saccharomyces cerevisiae.

Two signals activate meiosis in yeast: starvation and expression of the a1 and alpha 2 products of the mating-type locus. Prior studies suggest that these signals stimulate expression of an activator of meiosis, the IME1 (inducer of meiosis) product. We have cloned a gene, IME2, with properties similar to those of IME1: both genes are required for meiosis, and both RNAs are induced in meiotic cells. Elevated dosage of IME1 or IME2 stimulates the meiotic recombination pathway without starvation; thus, the IME products may be part of the switch that activates meiosis. IME1 was found to be required for IME2 expression, and a multicopy IME2 plasmid permitted meiosis in an ime1 deletion mutant. Accordingly, we propose that the IME1 product stimulates meiosis mainly through activation of IME2 expression.     

The C-terminal region of the meiosis-specific protein kinase Ime2 mediates protein instability and is required for normal spore formation in budding yeast

The cyclin-dependent kinase Cdk1 and the related kinase Ime2 act in concert to trigger progression of the meiotic cell cycle in the yeast Saccharomyces cerevisiae. These kinases share several functions and substrates during meiosis, but their regulation seems to be clearly different. In contrast to Cdk1, no cyclin seems to be involved in the regulation of Ime2 activity. Ime2 is a highly unstable protein, and we aimed to elucidate the relevance of Ime2 instability. We first determined the sequence elements required for Ime2 instability by constructing a set of deletions in the IME2 gene. None of the small deletions in Ime2 affected its instability, but deletion of a 241 amino acid C-terminal region resulted in a highly stabilized protein. Thus, the C-terminal domain of Ime2 is important for mediating protein instability. The stabilized, truncated Ime2 protein is highly active in vivo. Replacement of the IME2 gene with the truncated IME2ΔC241 in diploid strains did not interfere with meiotic nuclear divisions, but caused abnormalities in spore formation, as manifested by the appearance of many asci with a reduced spore number such as triads and dyads. The truncated Ime2 caused a reduction of spore number in a dominant manner. We conclude that downregulation of Ime2 kinase activity mediated by the C-terminal domain is required for the efficient production of normal four-spore asci. Our data suggest a role for Ime2 in spore number control in S. cerevisiae.