Production of functional small interfering RNAs by human Dicer

While RNA interference (RNAi) functions as a potent antiviral innate immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double knockout human cells lacking both Dicer and PKR. Using these cells, which tolerate dsRNA expression, we show that mutant forms of human Dicer lacking the amino-terminal helicase domain can process dsRNAs to produce high levels of siRNAs that are readily detectable by Northern blot and that can effectively and specifically inhibit the expression of cognate mRNAs. However, even these more active Dicer mutants produce only modest levels of viral siRNAs in infected cells that are insufficient to inhibit viral replication. We conclude that the production of siRNAs from viral dsRNAs is likely inefficient due to the poor accessibility of viral dsRNAs to Dicer. Overall design: Small RNA profiling of Dicer-deficient human NoDice(4-25) and NoDice(4-25)?PKR cells expressing full-length (WT) human Dicer or N-terminal Dicer truncations (F1, N1, N3) and their ability to process endogenous, ectopically-expressed, and viral double-stranded RNAs into small interfering RNAs (siRNAs).