Abstract
Until recently, Argonaute 2 (AGO2) and other RNA factors were believed to be restricted to the cytoplasm of mammalian somatic cells. It is now becoming appreciated that RNAi factors can also be found in cell nuclei, but much remains to be learned about their transport, molecular recognition, and function. We find that siRNA-mediated reduction of AGO1 or AGO2 increases the proportion of AGO1 or AGO2 in cell nuclei. Inhibition of AGO1 expression led to increased AGO2 levels, while knockdown of AGO2 led to increased levels of AGO1. Blocking AGO1, AGO2, or TRBP expression changed expression levels and nuclear distribution of RNAi factors Dicer, TNRC6A (GW182), and TRBP. These data reveal the expression of RNAi proteins is mutually dependent and that perturbation can affect subcellular distribution of those factors inside cells.
Highlights
Effects of small RNAs on silencing transcription were first noted by Morris and colleagues in 20047
Given the potential for nuclear RNAi to have a profound effect on gene regulation and reports of nuclear RNAi in other classes of organism[23,24,25], it may appear surprising that the phenomenon has not been explored more widely in mammalian cells
By contrast to reduced levels of TRBP, Dicer, and TRNC6A, silencing AGO1 led to ~1.4 fold increase in Argonaute 2 (AGO2) in both nuclear and cytoplasmic fractions (Fig. 2A,C,D) A similar increase in AGO2 levels was observed in MDA-MB-453 cells (Figure S2A)
Summary
Given the potential for nuclear RNAi to have a profound effect on gene regulation and reports of nuclear RNAi in other classes of organism[23,24,25], it may appear surprising that the phenomenon has not been explored more widely in mammalian cells. Several reports have described the presence of RNAi factors inside mammalian cell nuclei[30,31,32] consistent with the observations that RNA and RNAi factors influence classical nuclear processes such as splicing and transcription. Our data reveal that each AGO variant is retained in the cell nuclei relative to the cytoplasm after the depletion of each AGO, while cellular levels of AGO1/AGO2 are regulated in a compensatory manner. These data have implications for experiments investigating functions of nuclear AGO proteins and suggest that cells may have mechanisms that maintain pools of nuclear AGO proteins
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