1. Identification of the translational regulated LBP pathway in Gonyaulax Polyedra Liwei Fan MCB 186

2. Why do we use Gonyaulax Polyedra? Single cell Easily observable circadian regulated cellular processes such as the glows and flashes during night Drawbacks: it is a solid rock, very hard to perform traditional transgenic techniques on it.

3. Circadian regulation of bioluminescence in Gonyaulax involves translational control The enzyme: Luciferase (LCF) The substrate: Lucifercin The stabilizer: Luciferin Binding Protein (LBP) LCF and LBP are found to be translational controlled LBP mRNA level remains constant, whereas the LBP synthesis rate (Pulse labeling) and LBP level are rhythmically expressed Morse, D; Milos, P; Roux, E; Hastings, W : Circadian regulation of bioluminescence in Gonyaulax involves translational control, Figure 7. Cell biology. 86: 172-172, 1989

4. The proposed mechanism of the translational control of the LBP The significance of the 3’ untranslated region A dimer protein was detected to bind specifically to the 3’ UTR The protein’s binding activity cycles on a daily basis Max: day Min: night Seven UG repeats (important!) What could be the role(s) of this unknown protein : Repressor How did we find the protein binding to the 3’ UTR? Radioactive labeled transcript that covers the 3’ UTR was incubated with crude extracts from both the light and dark periods Mittag, M; Lee, D; Hastings, W. : Circadian expression of the luciferin-binding protein correlates with the binding of a protein to the 3’ untranslated region of its mRNA, Figure 5. Biochemistry. 91: 5257-5261, 1994

5. The problem: Why does the CCTR binding activity vary? Circadian-Controlled Translational Regulator (CCTR) A few Hypothesis –A change in the amount of CCTR –Phosphorylation and dephosphorylation of protein –Dependent on the secondary structure of the mRNA Goal –Identification of the CCTR –Identification of the translational regulated LBP pathway in Gonyaulax Why do we care? –The CCTR mechanism is a new type of circadian controlled pathway –The CCTR binding to the seven UG repeats is part of other circadian regulated pathways, for example: nitrogen and carbon metabolism in Chlamydomonas reinhardtii

6. The experiment Step 1: Microarray Analysis of the existing cDNA library of Gonyaulax Polyedra –2111 genes have been identified, 433 are known based on homology to other species –2800 genes have been identified in the Dinoflagellate Pyrocystis lunula, which is around 50% of the genes in Pyrocystis lunula Microarray will be performed at different circadian times –Dim light –0.5-2 ratio, separated by 12 hours ct Genes that exhibit circadian rhythmicity will be further examined subjected to phase shifts via light exposure –Light entrains the luminescence rhythm in Gonyaulax Polyedra –White light pulse and measurement of the level of mRNA of interest compared to control in constant dim light

7. The experiment Now we have identified genes that can respond to phase shift to light exposure –In pyrocystis lunula, we have identified 30 out of 2800 genes. Thus we are expecting 22-23 of such genes in Gonyaulax Polyedra –Since bioluminescence of Gonyaulax Polyedra is entrained to light, some of these genes will be involved in the translational regulated LBP pathway But which ones?

8. Proteins that affect the circadian rhythm in Gonyaulax Polyedra Staurosporine, 6-DMAP –Protein Kinase Inhibitor –It lengthens the FRP of G. Polyedra –Staurosporine is more specific than 6-DMAP In Vitro assay of the binding activity of these two kinase inhibitors to the protein product of the mRNA identified - Westernblot - Some of the proteins binding to these two Inhibitors will be involved in the LBP translational control pathway - More than one? - But proteins not affected by these two kinases can also play part in the pathway Comolli, J; Taylor, W; Hastings, W. : An Inhibitor of Protein Phosphorylation Stops the Circadian Oscillator and Blocks Light-Induced Phase Shifting in Gonyaulax polyedra, Figure 2B. Journal of Biological Rhythms, 9, 1: 13-26, 1994

9. Chlamydomonas reinhardtii CHLAMY1 = C1 + C3, binds to UG repeats and represses translation activity The sequence of this protein complex has been identified Homolog to the unknown CCTR in G. Polyedra? C1 and C3 are at constant level during circadian cycle C1 is sequestered by a large protein complex when not bound to the UG repeats on RNA Unlike G. Polyedra, genetic transformation can be easily performed on C. Reinhardtii CHLAMY1 and CCTR (G. Polyedra) controlled pathway in these two species can be seen as homologous

10. Searching for the homolog Confirmation that CCTR in G.Polyedra is indeed homologous to CHLAMY –In vitro assay of the CCTR with C1 and C3 antibodies Search for homologous circadian genes of translational controlled LBP in C. Reinhardtii The potential genes involved in the LBP translational control can be mapped to homologous genes in C.Reinhardtii that construct the same circadian regulated pathway (but different effects) as the translational controlled LBP pathway Gene transformation can be performed to identify the roles of these homologous genes in C. Reinhardtii –Fluorescent tagging with GFP, YFP, RFP to assess the identify of the protein complex binding to the C1 protein

11. One possible pathway of the translational regulation of LBP in G.Polyedra