Deamination of adenosines in extracellular RNA spontaneously internalized by human cells.
Ribonucleic acids circulating in mammalian extracellular fluids as well as RNAs accumulating in culture medium condensed by mammalian cells are internalized by acceptor cells and distributed among cellular compartments. The internalized RNA can be involved in the induction and regulation of cellular processes as guide or signaling molecules. The internalization of RNA may be accompanied by covalent modifications influencing the stability and functionality of this RNA. To analyze nucleotide modifications introduced by human cells in internalized extracellular RNA, 5.8S rRNA of S. cerevisiae was used. It was shown that 5.8S rRNA of S. cerevisiae is captured by human adenocarcinoma MCF-7 cells from culture medium and delivered to cytoplasm and nuclei. Most of the internalized RNA was hydrolyzed to mono- and oligonucleotides. Full-length RNA uptake was detected by RT-PCR in the cytoplasm and in nuclei after the pulse addition of RNA to the culture medium. 5'-Inosine was the only detectable modified nucleotide in the hydrolysate of cell-internalized RNAs. Consequently, extracellular RNAs entering human cells were subjected to partial adenosine deamination. Sequencing of cDNA confirmed that full-length extracellular RNA that accumulated in the nucleus, but not in the cytoplasm, was partially edited by adenosine deaminases. The deamination revealed in nuclear-stored, full-length RNA was site-specific. Adenosines edited in S. cerevisiae 5.8S rRNA are stack-closing A-U pairs A53 and A96, as well as unpaired A44 and A65. Our data emphasize the participation of adenosine deaminases in capturing and intracellular trafficking of internalized RNAs.
Institute of Chemical Biology and Fundamental Medicine, Siberian Division, Russian Academy of Sciences, Novosibirsk, Russia. kuligina@niboch.nsc.ru
Elena V
EV
Kuligina
Oksana V
OV
Vratskih
Dmitry V
DV
Semenov
Vera A
VA
Matveeva
Vladimir A
VA
Richter
D000241:Adenosine / Q000737:chemistry / Q000378:metabolism*; D000243:Adenosine Deaminase / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017393:RNA Editing; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012340:RNA, Ribosomal, 5.8S / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012331:RNA, Fungal; D012340:RNA, Ribosomal, 5.8S; D000243:Adenosine Deaminase; D000241:Adenosine