Primers used for the qRT-PCR.

<div> Clb1 and Clb2 are functionally redundant B-type cyclins, and the <i>clb1Δ clb2Δ</i> double mutant is lethal. In normal mitotic growth, Clb2 plays the central role in the G2-M progression. We previously demonstrated that the RNA-binding protein Puf5 positively regulates <i>CLB1</i> expression by downregulating expression of the repressor Ixr1. The decreased expression of <i>CLB1</i> by the <i>puf5Δ</i> mutation caused a severe growth defect of the <i>puf5Δ clb2Δ</i> double mutant. On the contrary, <i>CLB2</i> expression was unchanged between wild-type strain and <i>puf5Δ</i> mutant in unsynchronized cultures, and the <i>puf5Δ clb1Δ</i> double mutant did not show growth retardation. Therefore, we assumed that <i>CLB1</i> is the main target of Puf5 in the previous study. However, considering that <i>CLB1</i> and <i>CLB2</i> reportedly undergo a similar expression pattern during the cell cycle, we re-examined <i>CLB2</i> expression in the <i>puf5Δ</i> mutant in cell cycle-synchronized cultures and found that <i>CLB2</i> expression was decreased in the <i>puf5Δ</i> mutant strain. Deletion of <i>IXR1</i> restored the decreased expression of <i>CLB2</i> caused by the <i>puf5Δ</i> mutation. Moreover, we clarified that the decreased expression of <i>CLB2</i> caused by the <i>puf5Δ</i> mutation resulted in the growth defect in the S-phase cyclin deficient condition: the <i>puf5Δ clb1Δ clb5Δ clb6Δ</i> quadruple mutant grew worse than <i>clb1Δ clb5Δ clb6Δ</i> triple mutant, and the slow growth of the <i>puf5Δ clb1Δ clb5Δ clb6Δ</i> quadruple mutant was suppressed by <i>CLB2</i> overexpression. Moreover, the <i>ixr1Δ</i> mutation is known to be synthetically lethal with deletion of the <i>DUN1</i> gene encoding the checkpoint kinase. We found that the <i>clb2Δ</i> mutation restored the lethality of <i>ixr1Δ dun1Δ</i> double mutant. Our results suggest that Puf5 and Ixr1 regulate the cell cycle-specific expression of both <i>CLB1</i> and <i>CLB2</i>, that Clb5 and Clb6 have overlapping roles with Clb1 and Clb2, and that the regulation of <i>CLB1</i> and <i>CLB2</i> expression by Puf5 and Ixr1 is related to the function of Dun1 kinase. </div>