Gene silencing in mammalian cells by preformed small RNA duplexes.
Small interfering RNAs (siRNAs) mediate RNA interference (RNAi), a process in which target mRNAs are degraded. Here, we have investigated the efficacy of preformed siRNAs to modulate the expression of protein kinase Calpha (PKCalpha) and green fluorescent protein (GFP) in mammalian cells. We show that specific inhibition of PKCalpha and GFP can be achieved by in vitro transcribed siRNAs. Interestingly, a transcript harboring two self-complementary siRNAs interrupted by a single-stranded loop region inhibited both PKCalpha and GFP gene expression. These results suggest that the long transcript is processed by single-stranded ribonucleases and/or other proteins into two functional siRNAs. Incubation of the in vitro transcribed bispecific siRNA with protein extracts from HEK 293T cells yielded RNA duplexes similar to the synthetic single siRNA. Taken together, the present data indicate that in vitro transcribed siRNA can be useful for silencing gene expression. Additionally, bi- and perhaps poly-siRNAs may be expressed and processed in mammalian cells.
Leirdal
Marianne
M
Department of Immunology, Molecular Medicine Group, The Norwegian Radium Hospital, Montebello, Oslo N-0310, Norway.
Sioud
Mouldy
M
D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D005434:Flow Cytometry; D020868:Gene Silencing; D049452:Green Fluorescent Proteins; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D008164:Luminescent Proteins; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D049452:Green Fluorescent Proteins; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha