RESUMEN
In this study we obtained and characterized the recombinant analogue of multifunctional nucleolar phosphoprotein nucleophosmin 1 (NPM1) involved in crucial cellular processes such as transcription, reparation and mitosis. The influence ofnucleophosmin 1 on extrcellular RNAs accumulation in human adenocarcinoma cells MCF-7 was analyzed. It was found that incubation of AluY RNA (n > 300 nt), U24 snoRNA analogues (n ~ 80 nt) with Npm1-His6 resulted in RNA-protein non-covalent complexes formation, but not in case of the short oligoribonucleotide (n = 22 nt). It was shown that interaction of AluY RNA analogue with Npm1-His6 significantly increases transfection efficacy of the RNA into MCF-7 human cells. Altogether, these data allow us to conclude, that nucleophosmin 1 not only binds RNA with complex secondary structure, but also promotes uptake and internalization of such RNA by human cells.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN/metabolismo , Transfección/métodos , Escherichia coli/genética , Humanos , Células MCF-7/efectos de los fármacos , Células MCF-7/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/farmacología , Nucleofosmina , Ingeniería de Proteínas/métodos , ARN/química , ARN/farmacocinética , ARN Nucleolar Pequeño/farmacocinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologíaRESUMEN
11% of the human genome is composed of Alu-retrotransposons, whose transcription by RNA polymerase III (Pol III) leads to the accumulation of several hundreds to thousands of Alu-RNA copies in the cytoplasm. Expression of Alu-RNA Pol III is significantly increased at various levels of stress, and the increase in the Alu-RNA level is accompanied by a suppression of proliferation, a decrease in viability, and induction of apoptotic processes in human cells. However, the question about the biological functions of Pol III Alu-transcripts, as well as their mechanism of action, remains open. In this work, analogues of Alu-RNA and its evolutionary ancestor, 7SL RNA, were synthesized. Transfection of human breast adenocarcinoma MCF-7 cells with the Alu-RNA and 7SL RNA analogues is accompanied by a decrease in viability and by induction of proapoptotic changes in these cells. The analysis of the combined action of these analogues and actinomycin D or tamoxifen revealed that the decreased viability of MCF-7 cells transfected with Alu-RNA and 7SL RNA was due to the modulation of transcription. A whole transcriptome analysis of gene expression revealed that increased gene expression of the transcription regulator NUPR1 (p8), as well as the transcription factor DDIT3 (CHOP), occurs under the action of both the Alu- and 7SL RNA analogues on MCF-7 cells. It has been concluded that induction of proapoptotic changes in human cells under the influence of the Alu-RNA and 7SL RNA analogues is related to the transcriptional activation of the genes of cellular stress factors, including the endoplasmic reticulum stress response factors.