Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.
Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics.
Paul
Cynthia P
CP
Department of Biological Chemistry, University of Michigan, Ann Arbor, 48109, USA.
D001483:Base Sequence; D017403:In Situ Hybridization; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism*
D034741:RNA, Small Interfering; D012320:RNA Polymerase III