60ª Reunião Anual da SBPC

INTRODUÇÃO:

The cytoplasmatic and nuclear protein Ki-1/57 was firstly identified in malignant Hodgkin’s and Sternberg-Reed cells in Hodgkin’s lymphoma. Further studies have revealed that Ki-1/57 is phosphorylated by protein kinase C upon PMA cellular activation, which redistributes it to the cytoplasm and abolishes its interaction with the scaffold/signaling protein RACK1. Several p53-associated proteins and p53 itself also have been described as Ki-1/57 interacting partners. Moreover, Ki-1/57 interacts with and is methylated by PRMT1, an arginine methyltransferase that frequently methylates several RGG box-containing RNA binding proteins. Ki-1/57 is a RGG-box containing protein and beyond the above cited interacting proteins, it was also found to interact with several RNA binding proteins in our previous yeast two-hybrid screenings. These observations raise several insights for the function of Ki-1/57 and its relationship with the RNA metabolism. In this work we present the interaction analysis of Ki-1/57 with RNA binding proteins and RNA, strengthening its involvement with the RNA functions. Moreover, we show our ongoing analysis of sub-cellular localization, which contributes to the unveiling of the Ki-1/57 function.

METODOLOGIA:

The pull down assays were done with the purified bacterial or baculoviral recombinant proteins. After the coupling of the bait proteins to sepharose beads and the subsequent incubation of the prey proteins, the samples were subjected to high stringent washes and loaded on SDS-PAGE gels for western blots. Immunoprecipitation experiments were performed incubating the total lisates of Hela cells, protein G-sepharose beads and the antibody of interest under agitation overnight. After several washes, the samples were loaded on SDS-PAGE gels for western blots. The electrophoretic mobility shifty assays were developed incubating the purified recombinant proteins with the ³²P radio-labeled homopolymers probes (poly-A, poly-C, poly-G and poly-U) on ice. After this, the samples were loaded on native polyacrilamide gels for the analysis of the protein-RNA complexes by autoradiography. The sub-cellular localizations were performed after the transfection of HEK 293 cells (~40h) with pEGFP-C vectors containing truncated constructs of Ki-1/57 by the DNA-calcium phosphate precipitation method. The methylation inhibition treatments (Adox, 20 µM) were done after 12h of transfection and lasted until the end of the transfection process.

RESULTADOS:

In the present work, we present the interaction of Ki-1/57 with the RNA binding proteins SFRS9, SF2p32, YB-1 and hnRNP Q. The protein-protein associations were confirmed by in vitro pull-down assays and in vivo co-immunoprecipitations from Hela cells extracts. The putative binding of Ki-1/57 to RNA was tested through electrophoretic mobility shifty assays. In a screening of homopolymers probes, we found that Ki-1/57 is able to directly bind to RNA poly-U. In mapping experiments, we observed that Ki-1/57 binds to poly-U mainly through its RGG box-containing C-terminal. When we analyzed the sub-cellular localization of several Ki-1/57 constructs fused to GFP, we observed that under the treatment with the methylation inhibitor Adox only the full-length Ki-1/57 and its construct (1-222)Ki-1/57 localized to different nuclear sub-structures, evidencing the dependence of the N-terminal to this localization. Investigating the identity of these nuclear bodies under Adox treatment, we verified a perfect co-localization to the nucleolus of the full-length and the N-terminal Ki-1/57 constructs. A partial co-localization to speckles was also observed, suggesting a possible association with this sub-compartment in the nucleus.

CONCLUSÕES:

Taken as a whole, our findings pointed that Ki-1/57 may be a novel protein involved with the RNA functions. We confirmed the association of Ki-1/57 with several RNA binding proteins in vitro and in vivo. We found that Ki-1/57 is able to directly bind to RNA poly-U in a selective manner and mapped its C-terminal as involved with this binding. Our fluorescence microscopy analysis of cells under Adox treatment revealed that the Ki-1/57 localization to nuclear bodies is dependent of its N-terminal region. Further co-localization experiments showed that the full-length Ki-1/57 and its N-terminal (1-155)Ki-1/57 constructs localize to the nucleolus and possibly with speckles, as suggested by a partial co-localization with this nuclear body. Further assays are necessary to unveil the exact functional role of Ki-1/57 with the RNA functions.

Instituição de fomento: FAPESP, CNPq and LNLS

Palavras-chave: yeast two-hybrid system, RNA binding proteins, nuclear bodies

E-mail para contato: gbressan@lnls.br