Partial reconstitution of the RNAi response in human cells using Drosophila gene products.
While mammalian somatic cells are incapable of mounting an effective RNA interference (RNAi) response to viral infections, plants and invertebrates are able to generate high levels of viral short interfering RNAs (siRNAs) that can control many infections. In Drosophila, the RNAi response is mediated by the Dicer 2 enzyme (dDcr2) acting in concert with two cofactors called Loqs-PD and R2D2. To examine whether a functional RNAi response could be mounted in human somatic cells, we expressed dDcr2, in the presence or absence of Loqs-PD and/or R2D2, in a previously described human cell line, NoDice/ΔPKR, that lacks functional forms of human Dicer (hDcr) and PKR. We observed significant production of ∼21-nt long siRNAs, derived from a cotransfected double stranded RNA (dsRNA) expression vector, that were loaded into the human RNA-induced silencing complex (RISC) and were able to significantly reduce the expression of a cognate indicator gene. Surprisingly, dDcr2 was able to produce siRNAs even in the absence of Loqs-PD, which is thought to be required for dsRNA cleavage by dDcr2. This result may be explained by our finding that dDcr2 is able to bind the human Loqs-PD homolog TRBP when expressed in human cells in the absence of Loqs-PD. We conclude that it is possible to at least partially rescue the ability of mammalian somatic cells to express functional siRNAs using gene products of invertebrate origin.
Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Edward M
EM
Kennedy
Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Anand V R
AV
Kornepati
Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Hal P
HP
Bogerd
Department of Molecular Genetics and Microbiology and Center for Virology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Bryan R
BR
Cullen
D000818:Animals; D060846:Cell Engineering; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000172:deficiency / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D056918:Nuclear Receptor Coactivators / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D020365:RNA Helicases / Q000235:genetics* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000096:biosynthesis / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000096:biosynthesis / Q000235:genetics; D043244:Ribonuclease III / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D019076:Transgenes; D019892:eIF-2 Kinase / Q000172:deficiency / Q000235:genetics
D029721:Drosophila Proteins; C503213:Loqs protein, Drosophila; C486559:NCOA6 protein, human; D056918:Nuclear Receptor Coactivators; C478068:R2D2 protein, Drosophila; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; C000602769:EIF2AK2 protein, human; D019892:eIF-2 Kinase; C478069:DCR-2 protein, Drosophila; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases
Dicer; Drosophila; RNA interference; small interfering RNAs