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Effect of recombinant nucleophosmin 1 analogue on artificial RNA internalization into human adenocarcinoma MCF-7 cells

Abstract

In this study we obtained and characterized the recombinant analogue (Npm1-His6) of multifunctional nucleolar phosphoprotein nucleophosmin 1 (NPM1) involved in crucial cellular processes such as transcription, reparation, and mitosis. The interaction of Npm1-His6 with artificial noncoding RNAs in vitro and its influence on extracellular RNA accumulation in human adenocarcinoma MCF-7 cells were analyzed. It was found that the incubation of Npm1-His6 with AluY RNA (n > 300 nt) or U24 snoRNA analogues (n ∼ 80 nt) but not with a short oligoribonucleotide (n = 22 nt) resulted in the formation of the RNA-protein non-covalent complexes. Recombinant protein Npm1-His6 was shown to increase the transfection efficacy of RNA with a complex secondary structure into MCF-7 human cells. The results allow us to conclude that nucleophosmin 1 not only binds RNAs with complex secondary structure but also promotes the uptake and internalization of these RNAs by human cells.

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Abbreviations

HPRT:

gene, which encodes hypoxanthine-guanine phosphoribosyl transferase

IMDM:

Iskov’s modified Dulbecco’s medium

MCF-7:

human breast adenocarcinoma cell line

miR:

microRNA

NPM1:

nucleophosmin 1

Npm1-His6 :

recombinant analogue of human nucleophosmin 1

VLP:

virus-like particles of K2 killer yeast strain of S. cerevisiae Y448

MTT:

3-(4,5-dimethyl-2-thiasolyl)-2,5-diphenyl-2H-tetrazolium bromide

snoRNA:

small nucleolar RNA

NA:

nucleic acids

RT-PCR:

reverse transcription followed by polymerase chain reaction

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Correspondence to A. V. Savelyeva.

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Original Russian Text © A.V. Savelyeva, D.V. Semenov, G.A. Stepanov, D.N. Baryakin, E.V. Kuligina, I.V. Rabinov, O.A. Koval, V.A. Richter, 2014, published in Bioorganicheskaya Khimiya, 2014, Vol. 40, No. 1, pp. 55–63.

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Savelyeva, A.V., Semenov, D.V., Stepanov, G.A. et al. Effect of recombinant nucleophosmin 1 analogue on artificial RNA internalization into human adenocarcinoma MCF-7 cells. Russ J Bioorg Chem 40, 48–55 (2014). https://doi.org/10.1134/S1068162014010099

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  • DOI: https://doi.org/10.1134/S1068162014010099

Keywords

  • nucleophosmin 1
  • artificial RNA analogues
  • Alu RNA
  • box C/D RNA
  • RNA transfection into human cells
  • adenocarcinoma MCF-7 cells