Analysis of Est1-like proteins in chromosome segregation

Mechanisms controlling equal distribution of genetic information to daughter cells are fundamental for cell survival. A prerequisite for the faithful chromosome segregation is chromosome biorintation, a process during which sister chromatid kinetochores attach to microtubules radiating from the opposite spindle poles. Chromosome segregation is initiated through activation of the anaphase promoting complex (APC) only after all chromosomes biorient during metaphase. The APC plays a key role in the coordinating transition through mitosis and meiosis, as it couples chromatid separation with downregulation of the cyclin dependent kinase (CDK) activity. Our work in Arabidopsis identified a novel evolutionary conserved gene called EST1B, which appears to be an essential component of a novel and so far only poorly characterised mechanism controlling cell cycle progression during meiotic M phase. Inactivation of the gene results in a very unique phenotype characterized by anaphase arrest and failure to exit the second meiosis. This indicates that the EST1B protein is essential for coordination of meiotic chromosome segregation with cell cycle progression. The gene encodes a protein containing EST1 domain, which defines a conserved family of proteins found in all eukaryotes including human. The EST1 proteins are implicated in telomere maintenance and non-sense mediated RNA decay, but their role in other cellular processes is unknown. Here we propose to study a role of EST1 proteins in meiotic chromosome segregation using the model plant Arabidopsis thaliana and fission yeast Schizosaccharomyces pombe. In addition, we plan to investigate non-meiotic role of Arabidopsis EST1 proteins and perform functional analysis of the conserved EST1 domain. To understand the molecular mechanisms underlying function of the EST1B protein, we propose to perform transcriptome analysis and identify protein interaction partners. Because processes governing faithful chromosme segregation are evolutionary conserved, data obtained from the proposed study will likely to be applicable to other eukaryotes, including humans.

Nonsense mediated mRNA decay is an evolutionarily conserved RNA quality control mechanism that eliminates aberrant transcripts. This pathway is essential in higher eukaryotes and inactivation of the key NMD components is lethal in flies, mammals, and plants. However, how aberration in the NMD pathway translates into developmental defects and lethality is not well understood. In this project we examined the functions of the conserved NMD factor SMG7 in Arabidopsis. Plants carrying hypomorphic mutations in the SMG7 gene exhibit a range of seemingly pleiotropic phenotypes ranging from stunned growth to infertility. Interestingly, we were able to ascribe these phenotypes to defects in two very specific pathways: pathogen response and meiotic progression. We found that smg7 mutants exhibit hallmarks of a constitutively upregulated immune response and that genetic attenuation of this pathway fully suppresses all vegetative growth defects. Analysis of plants carrying mutations in other NMD genes (UPF1, UPF3) showed that the upregulation of the immune response is closely associated with NMD deficiency. In fact, the deregulated pathogen response is primarily responsible for lethality of a mutant that is fully deficient in NMD. This is a very significant finding suggesting that lethality of NMD deficient organisms is not caused by a pleiotropic deregulation of cellular metabolism, as is generally believed, but may rather be due to activation of a massive autoimmune response. We further discovered that infertility of smg7 mutants is caused by a specific arrest in meiotic progression in anaphase II. In contrast to vegetative defects, aberrant meiosis is specific to smg7 mutants and was not observed in other NMD mutants. Genetic analyses indicate that SMG7, together with another meiotic regulator, TDM1, act in the same pathway to facilitate exit from meiosis. We further demonstrate that the cyclin TAM is specifically expressed in meiosis I and has both stimulatory and inhibitory effects on progression to meiosis II. Based on these data we propose a model for regulation of meiotic progression in Arabidopsis. This model is important for designing experiments to decipher meiotic regulation in plants, which, in turn, may be important for development of new plant breeding strategies.