Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells.
RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.
Lee
Nan Sook
NS
Division of Molecular Medicine, Graduate School of Biological Sciences, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Kim
Daniel H
DH
Alluin
Jessica
J
Robbins
Marjorie
M
Gu
Shuo
S
Li
Haitang
H
Kim
James
J
Salvaterra
Paul M
PM
Rossi
John J
JJ
D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D034622:RNA Interference; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics*
D016337:RNA, Catalytic; D034741:RNA, Small Interfering; D012342:RNA, Small Nuclear; C405486:U6 small nuclear RNA; C419304:VA1-ribozyme chimera