Deadenylation of maternal mRNAs mediated by miR-427 in Xenopus laevis embryos

Abstract

We show that microRNA-427 (miR-427) mediates the rapid deadenylation of maternal mRNAs after the midblastula transition (MBT) of Xenopus laevis embryogenesis. By MBT, the stage when the embryonic cell cycle is remodeled and zygotic transcription of mRNAs is initiated, each embryo has accumulated approximately 10(9) molecules of miR-427 processed from multimeric pri-miR-427 transcripts synthesized after fertilization. We demonstrate that the maternal mRNAs for cyclins A1 and B2 each contain a single miR-427 target sequence, spanning less than 30 nucleotides, that is both necessary and sufficient for deadenylation, and that inactivation of miR-427 leads to stabilization of the mRNAs. Although this deadenylation normally takes place after MBT, exogenous miRNAs produced prematurely in vivo can promote deadenylation prior to MBT, indicating that turnover of the maternal mRNAs is limited by the amount of accumulated miR-427. Injected transcripts comprised solely of the cyclin mRNA 3' untranslated regions or bearing a 5' ApppG cap undergo deadenylation, showing that translation of the targeted RNA is not required. miR-427 is not unique in promoting deadenylation, as an unrelated miRNA, let-7, can substitute for miR-427 if the reporter RNA contains an appropriate let-7 target site. We propose that miR-427, like the orthologous miR-430 of zebrafish, functions to down-regulate expression of maternal mRNAs early in development.

FIGURE 1.

Biogenesis of miR-427 during early stages of Xenopus laevis embryogenesis. (A) Kinetics of accumulation and turnover of pri-miR-427, GS17 mRNA (an early indicator of zygotic transcription), and maternal cyclin B2 mRNA. The midblastula transition (MBT) and different stages of gastrulation (st 10, st 12) and hours post fertilization (p.f.) are shown above and below the autoradiograms of Northern blots containing one embryo-equivalent of total RNA per lane; the gel mobilities of ribosomal RNA and other size markers are indicated. (B) Northern blot analyses (1.5 embryo-equivalents of total RNA per lane) showing the precursor–product relationship of pre-miR-427 and mature miR-427 (top) and the absence of pre-miR-427 and miR-427 from embryos treated with α-amanitin (bottom). (*) Cross-hybridizing RNA that serves as loading control. (C) Northern blot analysis of pre-miR-427 and miR-427 in total RNAs from dissected pieces of stage 10.25 gastrula embryos. U6 RNA was also probed, for normalization. (T) Total; (Org) organizer half; (N-Org) nonorganizer half; (An) animal pole; (Vg) vegetal pole. (D) Southern blot of Xenopus genomic DNA (3.2 μg per lane) digested with the restriction enzymes indicated (BamH1, HindIII, BglII, EcoRI, EcoRV, PstI, StuI), showing the existence of a large number of identical miR-427-encoding regions that are devoid of BamH1 and HindIII cleavage sites. (E) Maps of representative EST clones (see Materials and Methods) used to deduce the structure of the 1.2-kb repeat unit in X. laevis genomic DNA (top) that encodes four (a–d) sequence variants of xla- miR-427 (bottom); (E) EcoRI cleavage sites; (S) StuI cleavage sites; (EV) EcoRV cleavage sites. The hybridization probe used in D is indicated.

FIGURE 2.

miR-427-dependent destabilization and deadenylation of cyclin B2 mRNA. (A) Nucleotide sequences in the cyclin B2 3′ UTR showing the predicted seed-match (shaded box) for miR-427 within a potential MRE₄₂₇, and potential miR-16 seed-matches (underlined). (B) Stabilization of cyclin B2 mRNA upon inactivation of miR-427. Northern blot analyses of endogenous cyclin B2 mRNA in early embryos that were untreated (top) or injected at the one-cell stage with 2′-OMe-antisense oligonucleotides against miR-427 (middle) or a let-7 control (bottom). (C) Impaired deadenylation of endogenous cyclin B2 mRNA upon inactivation of miR-427. (Top) Schematic of RNase H assay for determination of changes in poly(A) tail length, using a deoxyoligonucleotide that targets sequences ∼500 nt from the 3′ end of the mRNA. (Lower panels) Northern blot analyses of the 3′-terminal fragments of cyclin B2 mRNA generated by digestion with RNase H, showing the kinetics of deadenylation in the absence (top) or presence (bottom) of functional miR-427. Lanes A_n and A₀ show size markers generated by digestion of cyclin B2 mRNA of pre-MBT embryos with RNase H plus the deoxy-oligonucleotide, in the absence (A_n) or presence (A₀) of oligo-dT₁₅. (D) Northern blot analyses of pre-miR-427 and miR-427 in normal embryos (left) and in embryos injected with exogenous pre-miR-427 (right); a similar pattern of miRNA accumulation was seen when pre-miR-427_mut was injected. Analyses of RNAs from unfertilized eggs and one-cell (1 h p.f.) embryos (left panel) show the lack of pre-miR- and miR-427 in those cells. (E) Accelerated deadenylation of endogenous cyclin B2 mRNA upon premature expression of miR-427, but not miR-427_mut. Exogenous pre-miR-427 or pre-miR-427_mut was injected into one-cell embryos, and RNase H digested RNAs were analyzed as in C.

FIGURE 3.

Deadenylation of exogenous cyclin B2 reporter RNAs. (A) Schematic representation of the chimeric β-globin•cyclin B2 3′ UTR reporter mRNA (Gb•B2), indicating the seed-match for miR-427 (MRE), cytoplasmic polyadenylation elements (CPE), hexanucleotide polyadenylation signal (HEX), and pumilio binding element (PBE); the sequence of the PBE and substitution mutants of it are shown on the right. (B) Kinetics of polyadenylation and deadenylation of Gb•B2 reporter RNAs harboring wild-type (top) or mutant seed-matches (MRE_mut, bottom). Polyacrylamide gel analyses of ³²P-labeled reporter RNAs that were injected into two-cell embryos (∼2 h p.f.) and reisolated at the indicated times. Marker lanes (M) show the gel mobilities of the injected, nonpolyadenylated (A₀) transcripts. (C) PBE-dependent binding of Xenopus Pumilio protein (xPum). The electrophoretic mobility shift assays monitor in vitro complex formation between recombinant xPum (at 120 and 500 nM) and the indicated wild-type (wt) or substituted (PBE_mut, sub_61-88) 31-nt ³²P-labeled RNAs. (D) PBE-independent deadenylation. Kinetics of polyadenylation and deadenylation of Gb•B2 reporter RNAs containing wild-type or substituted PBE regions were analyzed as in B.

FIGURE 4.

Functions of MRE and PBE in poly(A) metabolism of cyclin B2 3′ UTR reporter RNAs. (A) Schematic representation of the cyclin B2 3′ UTR. (B) Kinetics of polyadenylation and deadenylation of wild-type RNA or substitution variants altering the MRE (MRE_mut) or PBE (sub_61–88). ³²P-labeled reporter RNAs were injected and assayed as in Figure 3B; samples were taken at 5, 6.5, 8, 10.5, 12.5, and ∼15 h p.f. A_S and A_L denote short and long poly(A) tail length, respectively (dotted lines). (C) Less extensive polyadenylation of the reporter RNA lacking the PBE. The pre-MBT samples (5 and 6.5 h p.f.) from the experiment shown in B were analyzed in the same gel, for direct comparison of the average poly(A) tail lengths (A_Ave, dotted lines). (D) Normal deadenylation kinetics of chimeric B2 3′ UTR reporter RNAs deleted of nucleotides 120–180 and the fused to hB4 3′ UTR sequences, which furnish CPE plus HEX functions. The wild-type (1–120 nt) and PBE or MRE substituted reporter RNAs were assayed as in B; samples were taken at 4.5, 6, 8, 9, 10.5, and 12 h p.f.

FIGURE 5.

MRE•miRNA base pairing in the deadenylation of cyclin A1 3′ UTR RNA. (A) Nucleotide sequences in the cyclin A1 3′ UTRs (top strand) showing the predicted seed-match (shaded box) and upstream 3′ UTR sequences that allow for functional pairing with miR-427 (bottom strand; cf. Fig. 6). The inactivating mutation of the MRE (MRE_mut) and the compensatory change in the seed sequence miR-427 (mut) are indicated. (B) Deadenylation of ³²P-labeled A1 3′ UTR reporter RNAs lacking a coding region and containing either a wild-type (wt) or mutant MRE (MRE_mut), or an ApppG- cap, to ensure lack of translation. (C) Sequences of wild-type and mutant forms of pre-miR-427. Nucleotide changes in pre-miR_mut (shaded) were designed to encode miR-427_mut with an altered seed sequence; (bold) mature miRNA sequences. (D) Rescue of MRE_mut by the compensatory mutation of the miR-427 seed sequence. Kinetics of polyadenylation and deadenylation of cyclin A1 3′ UTR reporter RNAs with wild-type (top panels) or mutant (bottom panels) seed-matches; the reporters were co-injected with exogenous pre-miRNAs generating either wild-type (left panels) or mutant (right panels) miR-427.

FIGURE 6.

Identification of regions in the cyclin A1 3′ UTR needed for deadenylation. (A) Nucleotide sequences of region 1–99 of the cyclin A1 3′ UTR and 11-nt substitutions (lowercase) that were tested for their effects on deadenylation. (B,C) Kinetics of deadenylation of A1 3′ UTR (1–99)•hB4 chimeric reporter RNAs with wild-type sequences (wt) or 11-nt substitutions (sub 1–9). Injected ³²P-labeled RNAs were analyzed as in Figure 3B, and samples were taken at 4, 5.5, 7.5, 8.5, 10.5, and 12 h p.f. (B) or 4, 5.5, 7.5, 8.5, 10.5, and 12 h p.f. (C). (D) A possible base-paired structure between miR-427 and nucleotides in region 12–22 (plus the seed-match) of wild-type A1 3′ UTR. Figure 5A shows a more likely alternative structure between miR-miR-427 and A1 3′ UTR that involves region 1–11.

FIGURE 7.

Compact structure and sequence-specific nature of MREs. (A) Sequences of the “minimal” cyclin A1 3′ UTR (1–33) capable of supporting deadenylation, and substitutions that inactivate the seed-match of MRE₄₂₇ (mut) or introduce an MRE_let-7. (B) Deadenylation of the wild-type A1 3′ UTR (1–33)•hB4 chimeric reporter RNA (left) is blocked upon substitution of MRE₄₂₇ (mut) and accelerated upon premature expression of miR-427 from co-injected pre-miR-427 (right). The samples were taken at 4, 6, 7.5, 8,5, 10.5, and 12.5 h p.f. (C) Deadenylation of the chimeric MRE_let-7 reporter RNA depends on expression of exogenous let-7 miRNA; pre-miRNAs that encode let-7 RNA or miR-427 were co-injected with the MRE_let-7 reporter, as indicated. The samples were taken at 4.5, 6, 7.5, 8.5, 9.5, and 12 h p.f. (D) The sequence of MRE_let-7 and its predicted base pairing with let-7 RNA (Mayr et al. 2007).