An Argonaute transports siRNAs from the cytoplasm to the nucleus

Abstract

Ribonucleoprotein complexes consisting of Argonaute-like proteins and small regulatory RNAs function in a wide range of biological processes. Many of these small regulatory RNAs are predicted to act, at least in part, within the nucleus. We conducted a genetic screen to identify factors essential for RNA interference (RNAi) in nuclei of Caenorhabditis elegans and identified the Argonaute protein NRDE-3. In the absence of small interfering RNAs (siRNAs), NRDE-3 resides in the cytoplasm. NRDE-3 binds siRNAs generated by RNA-dependent RNA polymerases acting on messenger RNA templates in the cytoplasm and redistributes to the nucleus. Nuclear redistribution of NRDE-3 requires a functional nuclear localization signal, is required for nuclear RNAi, and results in NRDE-3 association with nuclear-localized nascent transcripts. Thus, specific Argonaute proteins can transport specific classes of small regulatory RNAs to distinct cellular compartments to regulate gene expression.

Fig. 1

siRNA binding is necessary and sufficient for redistribution of NRDE-3 to the nucleus. (A) Predicted domain structure of R04A9.2. DNA lesions, identified in nrde-3 alleles, are indicated by arrows. (B) Sub-cellular localization of NRDE-3 correlates with NRDE-3-siRNA interactions. (top) Fluorescence microscopy visualizing GFP::NRDE-3 in seam cells of animals of the indicated genotypes (2). (bottom) FLAG::NRDE-3 co-immunoprecipitating RNAs were isolated, radiolabeled, and analyzed by PAGE (2). (C) siRNA binding is necessary to localize NRDE-3 to the nucleus. (top) NRDE-3 and NRDE-3(*PAZ) sub-cellular localization in seam cells and (bottom) co-precipitating endo siRNAs. (D) siRNA binding is sufficient for nuclear redistribution of NRDE-3. (top) GFP::NRDE-3 localization in seam cells and (bottom) FLAG::NRDE-3 co-precipitating exo siRNAs, in animals lacking endo siRNAs, 36 hours following exposure to control bacteria or dsRNA expressing bacteria (2).

Fig. 1

siRNA binding is necessary and sufficient for redistribution of NRDE-3 to the nucleus. (A) Predicted domain structure of R04A9.2. DNA lesions, identified in nrde-3 alleles, are indicated by arrows. (B) Sub-cellular localization of NRDE-3 correlates with NRDE-3-siRNA interactions. (top) Fluorescence microscopy visualizing GFP::NRDE-3 in seam cells of animals of the indicated genotypes (2). (bottom) FLAG::NRDE-3 co-immunoprecipitating RNAs were isolated, radiolabeled, and analyzed by PAGE (2). (C) siRNA binding is necessary to localize NRDE-3 to the nucleus. (top) NRDE-3 and NRDE-3(*PAZ) sub-cellular localization in seam cells and (bottom) co-precipitating endo siRNAs. (D) siRNA binding is sufficient for nuclear redistribution of NRDE-3. (top) GFP::NRDE-3 localization in seam cells and (bottom) FLAG::NRDE-3 co-precipitating exo siRNAs, in animals lacking endo siRNAs, 36 hours following exposure to control bacteria or dsRNA expressing bacteria (2).

Fig. 2

NRDE-3 is required for nuclear RNAi. (A) NRDE-3 is essential for silencing of the nuclear-localized pes-10::GFP RNA. (top) Light microscopy of ≈6 cell embryos subjected to in situ hybridization targeting the pes-10::gfp RNA +/− GFP dsRNA (2). (bottom) Fluorescence microscopy of ≈ 300 cell embryos +/− GFP RNAi. (B) NRDE-3 is required for RNAi-driven silencing of nuclear-localized lin-15 bicistronic RNA. qRT-PCR analysis of lin-15a/b pre-mRNA and lin-15b mRNA levels in animals +/− lin-15b dsRNA (n=4–8, +/− s.d.) (2). (C) NRDE-3 is not required for production of an endo siRNA. Total RNA isolated from animals of the indicated genotypes were subjected to Northern blot analysis detecting E01G4.5 endo siRNAs (2). (D) NRDE-3 is required for silencing of an endogenous pre-mRNA. qRT-PCR of E01G4.5 mRNA and E01G4.5 pre-mRNA levels from animals of the indicated genotypes (n=4, +/− s.d.) (2).

Fig. 3

NRDE-3 transports siRNAs from the cytoplasm to the nucleus: an obligatory step for nuclear RNAi. (A) NRDE-3 contains a functional NLS. Fluorescence microscopy of seams cells of animals expressing GFP::NRDE-3 and its variants (2). (B) Nuclear localization of NRDE-3 is required for nuclear RNAi. lir-1 RNAi-mediated lethality results from silencing of the nuclear-localized lir-1/lin-26 polycistronic RNA (2, 13). L1 animals of the indicated genotypes expressing the indicated NRDE-3 variants were fed bacteria expressing lir-1 dsRNA for 60 hours and scored for viability (n=3, +/− s.d.). (C) NRDE-3 associates with siRNAs in the cytoplasm. FLAG::NRDE-3 and FLAG::NRDE-3(*NLS) associated endo siRNAs were purified and labeled as described in Fig. 1B. (D) NRDE-3 associates with pre-mRNA in an RNAi-dependent manner. FLAG::NRDE-3 was immunoprecipitated from animals expressing indicated NRDE-3 variants after exposure to (+/−) lin-15b dsRNA. cDNAs generated from associating RNAs (n=4, +/− s.d.) were analyzed by qRT-PCR (2). Similar levels of NRDE-3 were immunoprecipitated in each experiment (not shown) and similar results were obtained following unc-40 RNAi (Fig. S5A).

Fig. 4

NRDE-3 associated siRNAs are generated by RdRPs acting upon cytoplasmic mRNA targets. (A) NRDE-3 associated siRNAs carry 5’ di- or tri-phosphates. FLAG::NRDE-3 associated endo siRNAs were treated (+/−) with guanylyl transferase and GTP and then detected as described in Fig. 1B. (B) Animals of indicated genotypes were fed (+/−) bacteria expressing unc-22 dsRNA for 24 hours and FLAG::NRDE-3-associated RNAs were detected as described in Fig. 1B. (C) eri-1(mg366) animals were fed bacteria expressing indicated dsRNAs. After 16 hours, GFP::NRDE-3 localization was assessed. Arrows indicate nuclear localization. (D) eri-1(mg366) animals were exposed to unc-40 dsRNA (targeted sequences indicated by vertical red lines) and FLAG::NRDE-3 associated siRNAs were cloned (2). Short black lines represent anti-sense unc-40 sequences identified.