SPO24 is a transcriptionally dynamic, small ORF-encoding locus required for efficient sporulation in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, meiosis and sporulation are highly regulated responses that are driven in part by changes in RNA expression. Alternative mRNA forms with extended 5' UTRs are atypical in S. cerevisiae, and 5' extensions with upstream open reading frames (uORFs) are even more unusual. Here we characterize the gene YPR036W-A, now renamed SPO24, which encodes a very small (67-amino-acid) protein. This gene gives rise to two mRNA forms: a shorter form throughout meiosis and a longer, 5'-extended form in mid-late meiosis. The latter form includes a uORF for a 14-amino-acid peptide (Spo24u14). Deletion of the downstream ORF (dORF) leads to sporulation defects and the appearance of pseudohyphae-like projections. Experiments with luciferase reporters indicate that the uORF does not downregulate dORF translation. The protein encoded by the dORF (Spo24d67) localizes to the prospore membrane and is differentially phosphorylated during meiosis. Transcription of the 5'-extended mRNA in mid-meiosis depends upon the presence of two middle sporulation elements (MSEs). Removal of the MSEs severely inhibits the mid-meiotic appearance of the 5'-extended mRNA and limits the ability of plasmid-borne SPO24 to rescue the sporulation defect of a spo24Δ mutant, suggesting that the 5'-extended mRNA is functionally important. These results reveal Spo24d67 as a sporulation-related factor that is encoded by a transcriptionally dynamic, uORF-containing locus.