Analysen des SR-Proteins Npl3 in der Translation und Charakterisierung von SR-Domänen-vermittelten Protein-Interaktionen von Npl3

Saccharomyces cerevisiae possesses only three SR-proteins in contrast to multi-cellular organisms. They are functioning as shuttling adaptor proteins in the nucleo-cytoplasmic transport of mRNAs. One important representative is Npl3 which is associated with actively translated mRNPs (messenger ribonucleoproteins). This study focused on a potential additional role of Npl3 besides its function in exporting mRNAs to the cytoplasm. The work contributed to the identification of Npl3 as a pre60S-export factor. Moreover, it was shown that Npl3 stays bound to the Rpl10-containing 60S-particle beyond the export and cytoplasmic maturation of pre60S-subunits. As a consequence, Npl3 has the capability to assist in translation by its association with translation competent 60S-particles. More specifically, truncating the SR-domain of Npl3 led to a reduced translation efficiency of yeast cells. In contrast, nuclear mRNA- and pre60S-export as well as the cytoplasmic maturation of 60S-particles were not substantially affected. Additionally, the results indicated that methylation in general or the phosphorylation site on position S411 in the SR-domain of Npl3 is not essential for translation. Rather, the truncation of the SR-domain resulted in a decreased binding of Npl3 to translation competent 60S-particles and a perturbance of monosome formation during translation initiation. This monosome formation defect is a result of inefficient binding of 60S-particles to mRNA-recruited 43S-initiation complexes. Different suppression experiments and genetic analyses in this work suggested a role of Npl3 in processes that are associated with the functions of Rpl10 and Fun12. Furthermore, an Npl3 homomer formation, dependent on the N-terminal region of the SR-domain, was demonstrated. Additionally, this specific part of the SR-domain of Npl3 was crucial for the binding of the nuclear import receptor Mtr10 supported by co-immunoprecipitation and immunofluorescence experiments. Moreover, the results indicate that the formation of Npl3-homomers might influence translation initiation positively. In summary, this work was able to assign Npl3 with its SR-domain an essential role for translation and suggested a function of Npl3 in the process of monosome formation during translation initiation as well as in the last cytoplasmic maturation step of pre60S-particles.