| It is well known that two size checkpoints operate in Chlamydomonas, however, the mechanisms to control cell size homeostasis are still unknown. A forward genetic screen based on insertional mutagenesis was used to identify new cell size checkpoint regulators. tny1-1 is an insertional mutant with a small-cell phenotype caused by checkpoint defects at Commitment and during S/M. Genetic epistasis experiments place TNY1 function upstream of MAT3/RB and the MAT3 kinase CDKG1. TNY1 encodes a putative RNA binding protein with two N-terminal RRM motifs and a low complexity C-terminus, a structure that is shared by several animal proteins in the hnRNP A/D, Musashi and DAZAP families. Complementation of tny1-1 by the wild type TNY1 gene indicates that the mutant is a loss of function allele and that TNY1 is a cell cycle repressor. The TNY1 message is cell cycle regulated with an expression peak during cell division (S/M). Intriguingly, the TNY1 protein expression pattern is also regulated, with a constant amount of TNY1 present per cell during the entire G1 period during which time cells in synchronous culture grow by around ten-fold in size. Consequently, the concentration of TNY1 within the cell steadily decreases during G1. This expression pattern suggests that TNY1 may be a titratable size threshold regulator whose function is to block cell cycle progression in cells that are below a minimum size for Commitment, and/or to drive exit from S/M when daughters arrive at the correct size. Interestingly, both CDKG1 and its partner CYCD2 have unusually long and GU-rich 3' untranslated regions (UTRs). Using North-Western assays, we found that TNY1 binds specifically to both CDKG1 and CYCD2 3' UTRs. Immunofluorescence localization experiments showed that TNY1 remains cytoplasmic throughout the cell cycle. These data, suggest that TNY1 levels may be used to control translation of CDKG1, CYCD2 and perhaps other genes whose products promote cell cycle progression. Experiments to test this model and to identify in vivo TNY1 targets are under way.
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