Figure 1.

Inhibition of miR-427-dependent deadenylation by exogeneous siRNA. (A) Schematic representation of the chimeric β-globin•cyclin B2 3′ UTR reporter mRNA (Gb•B2) indicating wild-type (wt) and inactivating mutant (mut) seed matches for miR-427 (MRE), cytoplasmic polyadenylation elements (CPE), and hexanucleotide polyadenylation signal (HEX). ³²P-labeled Gb•B2 reporter RNAs with wild-type (top, middle) or mutant MREs were injected alone or together with siRNA₄₂₇ (200 fmol per embryo) into one- to two-cell embryos [1.5–2 h post-fertilization), and polyadenylation and deadenylation were monitored over time by denaturing PAGE of total RNAs (one embryo equivalent per lane). Marker lanes (M) show the nonpolyadenylated reporter RNAs prior to injection, and stippled lines demarcate polyadenylated from deadenylated reporter RNAs. For nucleotide sequences of siRNA₄₂₇ (a miR-427 mimic), siRNA_mut (a variant designed to compensate for the seed match mutation in MREmut), and siRNA_NS (a nonspecific commercial siRNA), see the Materials and Methods. (B) Impaired deadenylation of endogenous cyclin B2 mRNAs independent of siRNA sequence. Kinetics of deadenylation in untreated embryos (Non-inj.) or embryos injected at the one-cell stage with siRNA₄₂₇ (left) or a non-specific siRNA_NS (right) (100 fmol per embryo) were monitored over time by Northern blot analyses of 3′-terminal fragments of cyclin B2 mRNA generated by digestion with RNase H. Marker lanes (M) show 3′-terminal fragments lacking poly(A) (A₀). (C) ³²P-labeled cyclin A1 3′ UTR reporter RNAs (top diagram) with wild-type or mutant miR427 target sites (MRE) were injected alone or together with the indicated siRNAs (200 fmol per embryo), and polyadenylation and deadenylation were monitored over time as in A.