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Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation.

Wada T, Hara M, Taneda T, Qingfu C, Takata R, Moro K, Takeda K, Kishimoto T, Handa H - Nucleic Acids Res. (2012)

Bottom Line: A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp.The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs.The MO removed the elongated poly(A) tail from maternal matured mRNA.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Biology, Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. tawada@tsurumi.yokohama-cu.ac.jp

ABSTRACT
Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3'-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.

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Injection of cdk9, but not cdk9m, MOs into embryos inhibits polyadenylation and translation of zcdk9 mRNA during early development. (A) Target sequence within the zcdk9 mRNA 3′-UTR and MO hybridization positions. There are five mismatches in cdk9m MO. (B) Inhibition of zcdk9 poly(A) tail elongation in cdk9 MO-injected embryos. Embryos were collected at the times indicated. Total RNA was extracted from untreated (WT), cdk9 MO-injected and cdk9m MO-injected embryos. PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Specific translation inhibition of zcdk9 mRNA by cdk9 MO, but not cdk9m MO. Embryos were collected at the indicated times. Western blotting was performed with extracts from untreated (lanes 1, 4, 7 and 10), cdk9 MO-injected (lanes 2, 5, 8 and 11) and cdk9m MO-injected embryos (lanes 3, 6, 9 and 12) by using the antibodies indicated. HeLa cell nuclear extracts (NEs) (lane 13) served as a control. Blots were probed with H-169, anti-zCdk9 and anti-actin. (D) The cdk9m MO (lanes 1 and 2), cdk9 MO (lanes 3 1nd 4) and 5′-cdk9 MO targeting a region around the first codon (lanes 5 and 6)-injected embryos were collected at 3 and 5 hpf. The extracts were subjected to western blotting by using antibodies indicated. (E and F) Reverse transcription using random (E) or oligo-dT primers (F), followed by PCR with primer sets for cdk9, biklf, tbp and actin, and analysis on a 2.2% agarose gel. Embryos were collected at the indicated times. Total RNA was extracted from untreated (WT: lanes 1–3), cdk9 MO-injected (cdk9 MO: lanes 4–6) and cdk9m MO-injected embryos (cdk9m MO: lanes 7–9).
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gks765-F1: Injection of cdk9, but not cdk9m, MOs into embryos inhibits polyadenylation and translation of zcdk9 mRNA during early development. (A) Target sequence within the zcdk9 mRNA 3′-UTR and MO hybridization positions. There are five mismatches in cdk9m MO. (B) Inhibition of zcdk9 poly(A) tail elongation in cdk9 MO-injected embryos. Embryos were collected at the times indicated. Total RNA was extracted from untreated (WT), cdk9 MO-injected and cdk9m MO-injected embryos. PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Specific translation inhibition of zcdk9 mRNA by cdk9 MO, but not cdk9m MO. Embryos were collected at the indicated times. Western blotting was performed with extracts from untreated (lanes 1, 4, 7 and 10), cdk9 MO-injected (lanes 2, 5, 8 and 11) and cdk9m MO-injected embryos (lanes 3, 6, 9 and 12) by using the antibodies indicated. HeLa cell nuclear extracts (NEs) (lane 13) served as a control. Blots were probed with H-169, anti-zCdk9 and anti-actin. (D) The cdk9m MO (lanes 1 and 2), cdk9 MO (lanes 3 1nd 4) and 5′-cdk9 MO targeting a region around the first codon (lanes 5 and 6)-injected embryos were collected at 3 and 5 hpf. The extracts were subjected to western blotting by using antibodies indicated. (E and F) Reverse transcription using random (E) or oligo-dT primers (F), followed by PCR with primer sets for cdk9, biklf, tbp and actin, and analysis on a 2.2% agarose gel. Embryos were collected at the indicated times. Total RNA was extracted from untreated (WT: lanes 1–3), cdk9 MO-injected (cdk9 MO: lanes 4–6) and cdk9m MO-injected embryos (cdk9m MO: lanes 7–9).

Mentions: We prepared cdk9 MO consisting of 25 bases that were completely complementary to the terminal sequence of the zcdk9 mRNA 3′-UTR (Figure 1A). To examine whether MOs against the 3′-UTR affect polyadenylation, a PAT assay (13) was performed, in which the PCR products reflect the poly(A) tail lengths present on a specific mRNA. When injected into fertilized embryos, cdk9 MO markedly reduced the production of slowly migrating bands compared to that of untreated embryos (WT), indicating that cdk9 MO inhibited polyadenylation (Figure 1B). The cdk9 MO also strongly reduced the amount of cdk9 PCR product, without affecting the products of tbp and cyclin B1. This result was specific for cdk9 MO, because embryos treated with cdk9m MO carrying 5-base mismatches acted no differently from untreated embryos (Figure 1B). These results suggest that cdk9 MO specifically affects cdk9 mRNA.Figure 1.


Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation.

Wada T, Hara M, Taneda T, Qingfu C, Takata R, Moro K, Takeda K, Kishimoto T, Handa H - Nucleic Acids Res. (2012)

Injection of cdk9, but not cdk9m, MOs into embryos inhibits polyadenylation and translation of zcdk9 mRNA during early development. (A) Target sequence within the zcdk9 mRNA 3′-UTR and MO hybridization positions. There are five mismatches in cdk9m MO. (B) Inhibition of zcdk9 poly(A) tail elongation in cdk9 MO-injected embryos. Embryos were collected at the times indicated. Total RNA was extracted from untreated (WT), cdk9 MO-injected and cdk9m MO-injected embryos. PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Specific translation inhibition of zcdk9 mRNA by cdk9 MO, but not cdk9m MO. Embryos were collected at the indicated times. Western blotting was performed with extracts from untreated (lanes 1, 4, 7 and 10), cdk9 MO-injected (lanes 2, 5, 8 and 11) and cdk9m MO-injected embryos (lanes 3, 6, 9 and 12) by using the antibodies indicated. HeLa cell nuclear extracts (NEs) (lane 13) served as a control. Blots were probed with H-169, anti-zCdk9 and anti-actin. (D) The cdk9m MO (lanes 1 and 2), cdk9 MO (lanes 3 1nd 4) and 5′-cdk9 MO targeting a region around the first codon (lanes 5 and 6)-injected embryos were collected at 3 and 5 hpf. The extracts were subjected to western blotting by using antibodies indicated. (E and F) Reverse transcription using random (E) or oligo-dT primers (F), followed by PCR with primer sets for cdk9, biklf, tbp and actin, and analysis on a 2.2% agarose gel. Embryos were collected at the indicated times. Total RNA was extracted from untreated (WT: lanes 1–3), cdk9 MO-injected (cdk9 MO: lanes 4–6) and cdk9m MO-injected embryos (cdk9m MO: lanes 7–9).
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gks765-F1: Injection of cdk9, but not cdk9m, MOs into embryos inhibits polyadenylation and translation of zcdk9 mRNA during early development. (A) Target sequence within the zcdk9 mRNA 3′-UTR and MO hybridization positions. There are five mismatches in cdk9m MO. (B) Inhibition of zcdk9 poly(A) tail elongation in cdk9 MO-injected embryos. Embryos were collected at the times indicated. Total RNA was extracted from untreated (WT), cdk9 MO-injected and cdk9m MO-injected embryos. PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Specific translation inhibition of zcdk9 mRNA by cdk9 MO, but not cdk9m MO. Embryos were collected at the indicated times. Western blotting was performed with extracts from untreated (lanes 1, 4, 7 and 10), cdk9 MO-injected (lanes 2, 5, 8 and 11) and cdk9m MO-injected embryos (lanes 3, 6, 9 and 12) by using the antibodies indicated. HeLa cell nuclear extracts (NEs) (lane 13) served as a control. Blots were probed with H-169, anti-zCdk9 and anti-actin. (D) The cdk9m MO (lanes 1 and 2), cdk9 MO (lanes 3 1nd 4) and 5′-cdk9 MO targeting a region around the first codon (lanes 5 and 6)-injected embryos were collected at 3 and 5 hpf. The extracts were subjected to western blotting by using antibodies indicated. (E and F) Reverse transcription using random (E) or oligo-dT primers (F), followed by PCR with primer sets for cdk9, biklf, tbp and actin, and analysis on a 2.2% agarose gel. Embryos were collected at the indicated times. Total RNA was extracted from untreated (WT: lanes 1–3), cdk9 MO-injected (cdk9 MO: lanes 4–6) and cdk9m MO-injected embryos (cdk9m MO: lanes 7–9).
Mentions: We prepared cdk9 MO consisting of 25 bases that were completely complementary to the terminal sequence of the zcdk9 mRNA 3′-UTR (Figure 1A). To examine whether MOs against the 3′-UTR affect polyadenylation, a PAT assay (13) was performed, in which the PCR products reflect the poly(A) tail lengths present on a specific mRNA. When injected into fertilized embryos, cdk9 MO markedly reduced the production of slowly migrating bands compared to that of untreated embryos (WT), indicating that cdk9 MO inhibited polyadenylation (Figure 1B). The cdk9 MO also strongly reduced the amount of cdk9 PCR product, without affecting the products of tbp and cyclin B1. This result was specific for cdk9 MO, because embryos treated with cdk9m MO carrying 5-base mismatches acted no differently from untreated embryos (Figure 1B). These results suggest that cdk9 MO specifically affects cdk9 mRNA.Figure 1.

Bottom Line: A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp.The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs.The MO removed the elongated poly(A) tail from maternal matured mRNA.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Cellular Biology, Department of Supramolecular Biology, Graduate School of Nanobioscience, Yokohama City University, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan. tawada@tsurumi.yokohama-cu.ac.jp

ABSTRACT
Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3'-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.

Show MeSH
Related in: MedlinePlus