Figure 5.

Lack of endogenous xlAgo2 catalytic activity in early embryos. (A) Inactivity of a coding region miR-427 target site. A ³²P-labeled Gb•B2 reporter RNA with a perfect match miR-427 target sequence within the β-globin coding region and an inactive MREmut in the 3′ UTR was injected into one- to two-cell embryos and analyzed as in Figure 1A. Note that the miR-427pm target site in the coding region does not elicit deadenylation at MBT. (B) siRNA₄₂₇-guided RNAi in preMBT embryos. The ³²P-labeled reporter RNA plus siRNA₄₂₇ (20 fmol per embryo) was injected alone or together with synthetic mRNAs encoding wild-type or mutant Myc-tagged hAgo2 and analyzed as in A. (C) Both 5′-terminal and 3′-terminal cleavage products were detected in the presence of hAgo2wt (Supplemental Fig. S3), and the extent of RNAi was quantified by PhosphorImager analyses. (D) siRNA_B2-guided RNAi of endogenous cyclin B2 mRNA. Synthetic mRNA encoding wild-type myc-tagged hAgo2 was injected alone or together with siRNA_B2 (30 fmol), and mRNA stability was monitored by Northern blot analysis of full-length cyclin B2 mRNA. The arrowhead indicates the 5′− terminal RNAi cleavage product. siRNA_B2 targets the 3′ UTR of X. laevis cyclin B2 mRNA (black bar in top schematic). (E) Ago2-mediated destabilization of the passenger strand of siRNA₄₂₇. Samples shown in B were analyzed by Northern blot hybridization using probes specific for the guide or passenger strand of siRNA₄₂₇. Marker lanes with one embryo equivalent of total RNA from noninjected stage 9.5 embryos (M) and siRNA₄₂₇ prior to injection (Inj.) show that the amount of siRNA₄₂₇ injected was comparable with that of endogenous miR-m27. The increase in guide strand hybridization signals at 5–6 h post-injection reflects the onset of endogenous miR-427 synthesis.