The ability to maintain cell size by balancing growth and division is found in all proliferating cells, but is a poorly understood process. Cell-size checkpoints that prevent division until cells have grown to a large enough size have been observed in many organisms, but the underlying mechanisms of size checkpoints have remained enigmatic. The long-term objectives of this research are to understand the molecular basis of cell-size checkpoint control in eukaryotic cells. The unicellular, eukaryotic green alga Chlamydomonas reinhardtii uses a variant cell cycle that partially uncouples growth from division, a property that makes its size checkpoint mechanism uniquely accessible. The central interpreter of cell size in Chlamydomonas is a Retinoblastoma (RB) tumor suppressor homologue, MATS, whose loss leads to lack of proper size checkpoint control and inappropriate cell division, similar to defects in human cancer cells. This proposal focuses on an RNA binding protein, TNY1, which regulates the size checkpoint pathway through MAT3/RB. TNY1 is related to the animal Musashi family of translational repressors that are involved in stem cell maintenance and cell fate determination. TNY1 is a negative regulator of the cell cycle that presumably binds mRNAs of key cell cycle genes and represses their translation.
The Aims of this proposal are: 1) To determine how TNY1 is regulated during the cell cycle and to determine whether it affects expression of a candidate target gene, CDKG1 that encodes a novel cyclin dependent kinase involved in size checkpoint control. 2) To comprehensively search for TNY1 RNA targets by identifying RNA sequences that interact with TNY1;to test the functional significance of such interactions;and pending results in Aim 1, to directly test binding of TNY1 to CDKG1 mRNA. 3) To independently identify rate limiting targets of TNY1 through genetic screens for tnyl suppressors, and to find TNY1 interacting proteins with a yeast two-hybrid screen. Relevance: Cancer is fundamentally a disease of mis-regulated growth and division, and the retinoblastoma tumor suppressor pathway is one of the key cellular guardians that prevent inappropriate proliferation and tumorigenesis. The work proposed here takes advantage of a simple unicellular model organism, Chlamydomonas, in order to dissect the regulation of the RB pathway. Work by the Umen laboratory has established a connection between a conserved RNA binding protein from the Musashi family of stem cell regulators, and cell division control through RB. The knowledge obtained from these studies may therefore be directly applicable to understanding basic principles that govern regulators that are central to human disease and stem cell maintenance.
