In vitro analyses of the production and activity of secondary small interfering RNAs in C. elegans

Abstract

In the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) play important roles as intermediates. Primary siRNAs are produced from trigger dsRNAs by an RNaseIII-related enzyme called Dicer; in some organisms, secondary siRNAs are also produced by processes involving RNA-dependent RNA polymerases (RdRPs), which act on target mRNAs. Using a cell-free assay system prepared from Caenorhabditis elegans, we analyzed the production and activity of secondary siRNAs. In this cell-free system, RdRP activity acts on mRNA-derived templates to produce small RNAs. The RRF-1 complex is predominantly responsible for the RdRP activity, and synthesizes secondary-type siRNA molecules in a Dicer-independent manner. Notably, secondary-type siRNAs induce a prominent Slicer activity to cleave target mRNAs far more effectively than primary-type siRNAs. An Argonaute protein, CSR-1, is responsible for the Slicer activity induced by secondary-type siRNAs. Secondary rather than primary siRNAs may play a major role in the destabilization of target transcripts during RNAi in C. elegans.

Figure 1

RdRP activity in a fraction of the cell lysate and its impairment by rrf-1 mutations. (A) Scheme for the preparation of cell lysates. Lysates from wild-type animals and viable mutants were prepared from large-scale cultures of mixed-stage populations. Lysates from sterile mutants were made from homozygotes at L4 or adult stages on a small scale, omitting the step indicated by the asterisk. (B) Aberrant mRNAs effectively induced RdRP activity in the cell-free system. In the presence of [α-³²P]UTP and other ribonucleotides, high-MW extracts pretreated with both MNase and DNase were reacted with the following templates (final 12.5 ng/μl): a dsRNA (542 bp), aberrant mRNA (1299 nt RNA lacking poly-A), or a normal mRNA (1359 nt). The RNA products were analyzed on a 15% sequencing gel. The electrophoretic data of template RNAs served as quality and loading controls. (C) The length of the small RNAs produced via the RdRP activity. (D) RdRP activity to produce the small RNAs was impaired in rrf-1(pk1417; fj18) mutants but was observed in the rde-4 mutant and wild-type (WT) animals. For the reactions to distinguish between RdRP activity and terminal transferase activity, 3′-O-methyl GTP was used instead of GTP. (E) RdRP activity to produce small RNAs was present in the dcr-1(ok247) mutant. High-MW extracts were prepared on a small scale from the dcr-1 mutant and wild-type animals. The template used for the RdRP assay in panels C, D and E was the aberrant mRNA. Ribosomal RNAs prepared from extracts served as loading controls.

Figure 2

RRF-1 interacts with DRH-3. (A) The GFP∷RRF-1 complex was immunopurified from lysates of transgenic animals. The proteins in the complex were resolved by SDS–PAGE and visualized by silver staining. Prominent protein bands were subjected to peptide mass fingerprinting analyses by MALDI-TOF mass spectrometry. The 130-kDa protein (p130) corresponded to DRH-3. The 75-kDa protein (asterisk) was Hsc70. (B) GFP∷RRF-1 immunoprecipitates did not contain a detectable amount of DCR-1. The immunoprecipitates and a portion of the input lysate were analyzed by immunoblotting using antibodies against GFP, DRH-3 and DCR-1. (C) RdRP activity to produce small RNAs was impaired in the drh-3(fj52) mutant. High-MW extracts were prepared on a small scale, and their RdRP activities towards an aberrant mRNA template were assayed. The 35-nt signal in lane 4 is background noise that was often detected in experiments with lysates prepared on a small scale.

Figure 3

The GFP∷RRF-1 complex has an RdRP activity to generate small RNAs. (A) Enzymatic activity of the GFP∷RRF-1 complex. Immunoprecipitations from the lysates of transgenic animals expressing GFP∷RRF-1 or wild-type animals were performed using the anti-GFP monoclonal antibody. Immunoprecipitates were treated with MNase to avoid contamination from endogenous RNAs and then washed with a buffer containing EGTA. Finally, the immunoprecipitates were reacted with an RNA template (393-nt mRNA), [α-³²P]CTP and other ribonucleotides. The RNA products were analyzed on a 15% sequencing gel. (B) Small RNAs generated by the RRF-1 complex were complementary to the template RNA. The GFP∷RRF-1 immunoprecipitates were reacted with an RNA template (1299-nt mRNA lacking poly-A) and radiolabeled ribonucleotides. Half of the RNA products were treated with a mixture of RNase A and T1 in the presence of 0.5 M NaCl. As a control, primary-type siRNAs produced from dsRNA by the Dicer activity in the RDE-4 complex were also analyzed. The RNA products were resolved on sequencing gels. (C) The RNA products described in B were resolved by 15% native PAGE. A chemically-synthesized 23-bp duplex siRNA (m) served as a size marker. (D) Small RNAs generated by the RdRP activity of GFP∷RRF-1 immunoprecipitates were found to be sensitive to treatment with vaccinia virus capping enzyme. M represents single-stranded RNA size markers.

Figure 4

Products and templates of the RdRP reactions. (A) Sequences of RdRP products. An RdRP reaction corresponding to lane 4 in Figure 3B was performed with non-radioactive nucleotides. The RdRP products were amplified by RT–PCR and cloned. (B) Long single-stranded RNAs are suitable templates for small-RNA synthesis by the RRF-1 complex. The GFP∷RRF-1 immunoprecipitates were reacted with the following RNA templates: primary-type siRNA (23-nt duplex), long dsRNA (542 bp), mRNA lacking poly-A (1299 nt) and normal mRNA (1359 nt).

Figure 5

Detection of mRNA cleavage (Slicer) activities induced by siRNAs. (A) Sequence-specific mRNA cleavage activities were induced by several types of siRNA in cell lysates from C. elegans. The cell lysates were reacted with a cap-radiolabeled mRNA and any of the following siRNAs: a monophosphorylated (5′ P) duplex (ds) siRNA, monophosphorylated single-stranded siRNAs and triphosphorylated (5′ PPP) single-stranded siRNA. The reaction products were analyzed on a 6% sequencing gel. ss, sense-oriented single strand; as, antisense-oriented single strand. (B) In the cell lysates, the triphosphorylated single-stranded siRNA mimicking secondary siRNAs was one order of magnitude more effective at inducing mRNA cleavage than the monophosphorylated single-stranded siRNA. (C) Target mRNA cleavages induced by several single-stranded siRNAs with different sequences. The siRNA no. 7 was used for the assays in panels A and B.

Figure 6

Similarities and differences in the Slicer activities induced by mono- and triphosphorylated siRNAs. (A) mRNA substrates to confirm the site of mRNA cleavage by Slicer activity. A 2-nt mismatch mutation was introduced into the mRNA site where cleavage was suggested by RT–PCR analysis. (B) mRNA carrying the 2-nt mismatch was not cleaved efficiently by the activities induced by any of the three types of siRNA. The abbreviations are the same as those described in the legend for Figure 5A. (C) Effect of ATP on the target mRNA destruction induced by several types of siRNA. 5′ phosphorylated siRNAs (5′ P or 5′ PPP) or 5′ non-phosphorylated siRNAs (5′ OH) were introduced into standard lysates containing the ATP regeneration system (lanes 1, 3, 5, 7 and 9) and ATP-depleted lysates (lanes 2, 4, 6, 8 and 10). (D) Gel-filtration analysis of Slicer factors that respond to single-stranded siRNAs. The standard lysate was fractionated into 30 fractions using a Superdex 200 (GE) column, and adjacent pairs were pooled into 15 samples. siRNAs and labeled mRNAs were reacted in each sample. The fractionation pattern of molecular weight markers is indicated by the arrowheads. Fractions 1, 2, 3 and 4 correspond to the void fraction (Vo).

Figure 7

CSR-1 is predominantly responsible for the Slicer activity induced by secondary-type siRNAs. (A) Single-stranded siRNAs properly induced target mRNA cleavages in cell lysates from rde-1, rrf-1, rde-3 and rde-4 mutants and those from wild-type animals. To confirm equality of the lysate concentration, the amount of GAPDH was monitored by immunoblotting. (B) The Slicer activity induced by a secondary-type siRNA bearing a 5′ triphosphorylated end was impaired in the csr-1(fj54) mutant. Cell lysates were prepared on a small scale from the csr-1 mutant and wild-type animals, and served for the Slicer assay. (C) Inhibition of Slicer activities by a 2′-O-methyl oligonucleotide. Reactions were performed with 100 nM secondary-type siRNAs and 400 nM 2′-O-methyl oligonucleotide complementary to siRNA no. 7. (D) CSR-1 interacted in vitro with the secondary-type siRNA. The siRNA/Slicer complex was captured by the 2′-O-methyl oligonucleotide mimicking the target mRNA, and was examined by immunoblotting. (E) Enzymatic activities of CSR-1 recombinant proteins. 6 × His∷MBP∷CSR-1 fusion proteins were expressed in bacteria and purified. The catalytic domain of 6 × His∷MBP∷CSR-1(D769A) was designed to have a substitution of a conserved aspartic acid by an alanine. Recombinant proteins (final 5.3 ng/μl) were incubated with mono- or triphosphorylated siRNAs and then reacted with a radiolabeled target mRNA.

Figure 8

Model for the RNAi pathway in C. elegans (see text).