Functional characterization of the nuclear basket tpr nucleoporins in the fission yeast

Resumen

Research performed over the last years has changed the view of NPCs as simple nucleo-cytoplasmic trafficking channels into a more comprehensive understanding of the multiples roles of the NPCs, which range from chromatin regulation to the maintenance of genome integrity. The most nucleoplasm-facing structure of the NPC is the nuclear basket. While in higher eukaryotes the main structural component of the nuclear basket is the translocated promoter region (TPR) nucleoporin, most yeasts possess two orthologs: Mlp1/Mlp2 in Saccharomyces cerevisiae and Nup211/Alm1 in Schizosaccharomyces pombe. Although it is known that most nuclear basket functions have been conserved along evolution, it remains unclear how TPR nucleoporins are assembled into the NPCs and the roles that they perform in the fission yeast. Previous data from our laboratory described that the absence of Alm1 leads to chromosome missegregation and altered kinetochore behaviour. In order to avoid erroneous microtubule-kinetochore attachments and chromosome segregation defects, it is required a proper centromere and kinetochore structural organization, which is regulated by proteasomal degradation. Proteasome is enriched in the nucleus, specially at the nuclear periphery, although how this localization is regulated and its biological implications are unclear. In the first part of this thesis, we have characterized the role of the nuclear basket component Alm1 in the spatial regulation of the proteasome, which is key for chromosome segregation through the regulation of the kinetochore homeostasis. The different steps of mRNA biogenesis, including transcription, processing, quality control and export are closely coordinated, and the nuclear basket has been proposed to act as a physical platform that couples such processes. nup211+ was previously described as required for mRNA export. However, it remains unknown its specific role in mRNA docking and export. In the second part of this thesis, we have characterized how the two TPR nucleoporins in the fission yeast, Nup211 and Alm1, are assembled into the nuclear basket and how they anchor to the NPC. Additionally, we have performed a genetic and functional analysis to dissect the functions of Nup211 and Alm1 in mRNA docking, quality control and export. Heat shock deeply compromise cell viability due to protein denaturation and aggregation. In order to ensure survival cells activate the evolutionary conserved heat shock response (HSR), which results in profound changes in mRNA metabolism and nuclear organization. How this switch is achieved is not fully understood. In the third part of this study, we have characterized how heat stress leads to the inhibition of bulk mRNA export and the arrest of cell growth, concomitant with the aggregation of NPC components, the mRNA processing and export machinery, cell cycle regulators, and protective chaperones and disaggregases into ring-like structures proximal to the nucleolus. We propose that these structures, named “nucleolar rings” (NuRs), are formed by the reversible aggregation of nuclear components, and constitute storage sites for those activities that are inhibited during HS and have to be protected in order to re-start cellular metabolism when normal conditions are restored.