Abstract
Execution of the meiotic and mitotic cell division programs requires distinct gene expression patterns. Unlike mitotic cells, meiotic cells reduce ploidy by following one round of DNA replication with two rounds of chromosome segregation (meiosis I and meiosis II). However, the mechanisms by which cells prevent DNA replication between meiosis I and meiosis II are not fully understood. Here, we show that transcriptional repression of two essential DNA replication genes, CDC6 and SLD2, is associated with production of shorter meiosis-specific RNAs containing the 3′ end of both genes. Despite the short CDC6 RNA coding for a short protein (Cdc6short), this protein is not essential for meiosis and it does not have either a positive or negative impact on DNA replication. Production of CDC6short mRNA does not require the upstream CDC6 promoter (PCDC6) and is not a processed form of the full-length RNA. Instead, CDC6short depends on transcription initiation from within the ORF upon repression of PCDC6. Finally, using CDC6 genes from related yeast, we show that repression of full-length CDC6 mRNA is evolutionarily conserved and that this repression is consistently associated with production of unique short CDC6 RNAs. Together, these data demonstrate that meiotic cells transcriptionally repress full-length CDC6 and SLD2, and that inactivation of PCDC6 results in heterogeneous transcription initiation from within the CDC6 ORF.
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Acknowledgements
We thank Luke Berchowitz for comments on the manuscript. We thank all members of the Bell Laboratory for helpful discussions. We thank John Diffley, Maitreya Dunham, and Mark Johnston for strains. DVP was supported in part by a NIH Pre-Doctoral Training Grant (GM007287). SPB is an investigator with the Howard Hughes Medical Institute.
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Fig. S1
Cells from Fig. 1 proceed synchronously through meiosis. (a) Cell-cycle stage quantification for Fig. 1a. (b) Only the full-length Sld2 protein isoform is detectable during meiosis (yDP336). Top: Sld2-13myc immunoblot during meiosis with protein samples run on a 12% SDS-PAGE gel. Size markers are indicated to the right of the gel. These are the same protein samples as those in Fig. 1b. Bottom: Cell-cycle stage quantification for samples used in this figure and Fig. 1b. (c) Cell-cycle stage quantification for Fig. 1c. (PNG 563 kb) (PNG 563 kb)
Fig. S2
Characterization of the CDC6short and SLD2short RNA’s and Cdc6short protein. (a) 5’ RACE of SLD2 shows heterogeneous 5’ ends during the meiotic divisions. Left: Agarose gel of PCR products. Portions that were gel purified for ligation into a cloning vector are denoted by colored boxes, along with the range of 5’ ends detected for each species. Right: Diagram of SLD2 promoter, ORF, and 13myc tag with the relative location of SLD2-specific primers for the RT reaction and PCR reactions. Colored vertical lines and numbers indicate the 5’ ends of individually sequenced clones from specific gel-purified sections. (b) Neither full-length Cdc6 nor Cdc6short depend on Met120, Met261, or Met267. Plasmids containing PCDC6-CDC6-3V5 with Met→Val start codon mutations were integrated into yeast, and the subsequent strains were assayed for the ability to produce Cdc6short during meiosis. CDC6Met-120-Val (yDP226, lanes 1-7); CDC6Met-261-Val (yDP228, lanes 8-14); CDC6Met-267-Val (yDP229, lanes 15-21); CDC6Met-261/267-Val (yDP230, lanes 22-28). The time after transfer into sporulation medium is indicated above each lane. Protein was detected by V5-immunoblot. (PNG 632 kb)
Fig. S3
Cdc6short does not form a stable complex with ORC, Cdt1, and Mcm2-7 in ATPγS. (a) WT Cdc6 can form the OCCM complex (lanes 2-6), whereas Cdc6short cannot form the OCCM complex (lanes 4-7) at all concentrations tested in ATPγS. Top: Total protein stain (Krypton stain). Bottom: FLAG-immunoblot to specifically examine Cdc6 vs Cdc6short, as well as Mcm3 and Orc1, all of which have a FLAG epitope tag. (PNG 296 kb)
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Phizicky, D.V., Bell, S.P. Transcriptional repression of CDC6 and SLD2 during meiosis is associated with production of short heterogeneous RNA isoforms. Chromosoma 127, 515–527 (2018). https://doi.org/10.1007/s00412-018-0681-x
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DOI: https://doi.org/10.1007/s00412-018-0681-x