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This was an exploratory one-year study to identify chemical regulators of the COP9 signalosome. Chemical Genetics uses small molecules to modify or disrupt the function of specific genes/proteins. This is in contrast to classical genetics, in which mutations disrupt the function of genes. The underlying concept is that the functions of most proteins can be altered by the binding of a chemical, which can be found by screening large libraries for compounds that specifically affect a biological, molecular or biochemical process. In addition to screens for chemicals which inhibit specific biological processes, chemical genetics can also be employed to find inhibitors of specific protein-protein interactions. Small molecules altering protein-protein interactions are valuable tools in probing protein-protein interactions. In this project, we aimed to identify chemicals that disrupt the COP9 signalosome. The CSN is an evolutionarily conserved eight-subunit protein complex whose most studied role is regulation of E3 ubiquitinligase activity. Mutants in subunits of the CSN undergo photomorphogenesis in darkness and accumulate high levels of pigments in both dark- and light-grown seedlings, and are defective in a wide range of important developmental and environmental-response pathways. Our working hypothesis was that specific molecules will interact with the CSN7 protein such that binding to its various interacting proteins will be inhibited. Such a molecule would inhibit either CSN assembly, or binding of CSN-interacting proteins, and thus specifically inhibit CSN function. We used an advanced chemical genetic screen for small-molecule-inhibitors of CSN7 protein-protein interactions. In our pilot study, following the screening of ~1200 unique compounds, we isolated four chemicals which reproducibly interfere with CSN7 binding to either CSN8 or CSN6.

The original working hypothesis of our proposal was that The “e” subunit of eIF3 has multiple functions from both within the nucleus and in the cytoplasm. Within this model, we further hypothesized that the “e” subunit of eIF3 functions in translation as a repressor. We proposed to test these hypotheses along the following specific aims: 1) Determine the subcellular localization of the interaction between eIF3e and other eIF3 subunits, or the COP9 signalosome. 2) Elucidate the biological significance of the varied subcellular localizations of eIF3e through generating Arabidopsis eIF3e alleles with altered subcellular localization. 3.) Purify different eIF3e complexes by tandem affinity purification (TAP). 4) Study the role of eIF3e in translational repression using both in vitro and in planta assays. eIF3 is an evolutionarily ancient and essential component of the translational apparatus in both the plant and animal kingdoms. eIF3 is the largest, and in some ways the most mysterious, of the translation factors. It is a multi-subunit protein complex that has a structural/scaffolding role in translation initiation. However, despite years of study, only recently have differential roles for eIF3 in the developmental regulation of translation been experimentally grounded. Furthermore, the roles of individual eIF3 subunits are not clear, and indeed some, such as the “e” subunit may have roles independent of translation initiation. The original three goals of the proposal were technically hampered by a finding that became evident during the course of the research – Any attempt to make transgenic plants that expressed eIF3e wt or eIF3e variants resulted in seedling lethality or seed inviability. That is, it was impossible to regenerate any transgenic plants that expressed eIF3e. We did manage to generate plants that expressed an inducible form of eIF3e. This also eventually led to lethality, but was very useful in elucidating the 4th goal of the research (Yahalom et al., 2008), where we showed, for the first time in any organism, that eIF3e has a repressory role in translation. In attempt to solve the expression problems, we also tried expression from the native promoter, and as such analyzed this promoter in transgenic plants (Epel, 2008). As such, several additional avenues were pursued. 1) We investigated protein-protein interactions of eIF3e (Paz-Aviram et al., 2008). 2) The results from goal #4 led to a novel hypothesis that the interaction of eIF3e and the CSN meets at the control of protein degradation of nascent proteins. In other words, that the block in translation seen in csn and eIF3e-overexpressing plants (Yahalom et al., 2008) leads to proteasome stress. Indeed we showed that both over expression of eIF3e and the csn mutants lead to the unfolded protein response. 3) We further investigated the role of an additional eIF3 subunit, eIF3h, in transalational regulation in the apical meristem (Zhou et al., 2009). Epel, A. (2008). Characterization of eIF3e in the model plant Arabidopsis thaliana. In Plant Sciences (Tel Aviv, Tel Aviv University). Paz-Aviram, T., Yahalom, A., and Chamovitz, D.A. (2008). Arabidopsis eIF3e interacts with subunits of the ribosome, Cop9 signalosome and proteasome. Plant Signaling and Behaviour 3, 409-411. Yahalom, A., Kim, T.H., Roy, B., Singer, R., von Arnim, A.G., and Chamovitz, D.A. (2008). Arabidopsis eIF3e is regulated by the COP9 signalosome and has an impact on development and protein translation. Plant J 53, 300-311. Zhou, F., Dunlap, J.R., and von Arnim, A.G. The translation initiation factor subunit eIF3h is .1 involved in Arabidopsis shoot apical meristem maintenance and auxin response. (submitted to Development).

Plants adjust their growth and development in a manner optimal for the prevailing light conditions. The molecular mechanisms by which light signals are transduced and integrated with other environmental and developmental signals are an area of intense research. (Batschauer, 1999; Quail, 2002) One paradigm emerging from this work is the interconnectedness of discrete physiological responses at the biochemical level, for instance, between auxin and light signaling (Colon-Carmona et al., 2000; Schwechheimer and Deng, 2001; Tian and Reed, 1999) and between light signaling and plant pathogen interactions (Azevedo et al., 2002; Liu et al., 2002). The COP9 signalosome (CSN) protein complex has a central role in the light control of plant development. Arabidopsis mutants that lack this complex develop photomorphogenically even in the absence of light signals (reviewed in (Karniol and Chamovitz, 2000; Schwechheimer and Deng, 2001). Thus the CSN was hypothesized to be a master repressor of photomorphogenesis in darkness, and light acts to bypass or eliminate this repression. However, the CSN regulates more than just photomorphogenesis as all mutants lacking this complex die near the end of seedling development. Moreover, an essentially identical complex was subsequently discovered in animals and yeast, organisms whose development is not light responsive, exemplifying how plant science can lead the way to exciting discoveries in biomedical model species (Chamovitz and Deng, 1995; Freilich et al., 1999; Maytal-Kivity et al., 2002; Mundt et al., 1999; Seeger et al., 1998; Wei et al., 1998). Our long-term objective is to determine mechanistically how the CSN controls plant development. We previously that this complex contains eight subunits (Karniol et al., 1998; Serino et al., 1999) and that the 27 ilia subunit is encoded by the FUS5/CSN7 locus (Karniol et al., 1999). The CSN7 subunit also has a role extraneous to the COP9 signalosome, and differential kinase activity has been implicated in regulating CSN7 and the COP9 signalosome (Karniol et al., 1999). In the present research, we further analyzed CSN7, both in terms of interacting proteins and in terms of kinases that act on CSN7. Furthermore we completed our analysis of the CSN in Arabidopsis by analyzing the remaining subunits. Outline of Original Objectives and Subsequent Modifications The general goal of the proposed research was to study the CSN7 (FUS5) subunit of the COP9 signalosome. To this end we specifically intended to: 1. Identify the residues of CSN7 that are phosphorylated. 2. Monitor the phosphorylation of CSN7 under different environmental conditions and under different genetic backgrounds. 3. Generate transgenic plants with altered CSN7 phosphorylation sites. 4. Purify CSN7 kinase from cauliflower. 5. Clone the Arabidopsis cDNA encoding CSN7 kinase 6. Isolate and characterize additional CSN7 interacting proteins. 7. Characterize the interaction of CSN7 and the COP9 signalosome with the HY5-COP1 transcriptional complex. Throughout the course of the research, emphasis shifted from studying CSN7 phosphorylation (Goals 1-3), to studying the CSN7 kinase (Goal 4 and 5), an in depth analysis of CSN7 interactions (Goal 6), and the study of additional CSN subunits. Goal 7 was also abandoned as no data was found to support this interaction.