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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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Specificity of polyadenylation inhibition by MO. (A) Sequences of cyclin B1 and B2 MOs. (B) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 2–4) or uninjected (lane 1). A PAT assay was performed with PAT primers for cdk9, cyclin B1 and cyclin B2. PCR using the actin primer set was performed with the cDNA produced in the PAT assay (actin). PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 1–5) or uninjected (lane 6). A PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. The PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases.
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gks765-F4: Specificity of polyadenylation inhibition by MO. (A) Sequences of cyclin B1 and B2 MOs. (B) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 2–4) or uninjected (lane 1). A PAT assay was performed with PAT primers for cdk9, cyclin B1 and cyclin B2. PCR using the actin primer set was performed with the cDNA produced in the PAT assay (actin). PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 1–5) or uninjected (lane 6). A PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. The PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases.

Mentions: To validate the effect of MO on polyadenylation, three kinds of gene-specific MOs were created, including cyclin B1 MO, cyclin B2 MO and tbp MO, in addition to cdk9 MO (Figure 4). To determine the MO specificity, the total RNA derived from 5 hpf embryos was examined, and a PAT assay was performed (Figure 4B and C). We tested a mixture of cyclin B1 MO plus cyclin B2 MO (Figure 4A and B). Zebrafish has the cyclin B orthologs B1 and B2, and their 3′-UTRs exhibit sequence similarity; indeed, there were 10-base homologies between cyclin B1 MO and cyclin B2 MO (Figure 4A). Cyclin B1 MO and cyclin B2 MO each affected its own target without oligonucleotides interference (Figure 4B). We also observed the specific action of MO when cdk9 MO and tbp MO were examined (Figure 4C). The effects of MO on the polyadenylation of mRNAs were confirmed by using RNA samples collected from 2 to 5 hpf embryos (Supplementary Figure S2A and S2B). In addition, each MO binds to a target 3′-UTR in a specific manner (Supplementary Figure S3). These results indicate that antisense MOs targeting the 3′-UTR of mRNAs affect polyadenylation in early zebrafish embryos in a specific manner.Figure 4.


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)

Specificity of polyadenylation inhibition by MO. (A) Sequences of cyclin B1 and B2 MOs. (B) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 2–4) or uninjected (lane 1). A PAT assay was performed with PAT primers for cdk9, cyclin B1 and cyclin B2. PCR using the actin primer set was performed with the cDNA produced in the PAT assay (actin). PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 1–5) or uninjected (lane 6). A PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. The PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3526265&req=5

gks765-F4: Specificity of polyadenylation inhibition by MO. (A) Sequences of cyclin B1 and B2 MOs. (B) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 2–4) or uninjected (lane 1). A PAT assay was performed with PAT primers for cdk9, cyclin B1 and cyclin B2. PCR using the actin primer set was performed with the cDNA produced in the PAT assay (actin). PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 1–5) or uninjected (lane 6). A PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. The PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases.
Mentions: To validate the effect of MO on polyadenylation, three kinds of gene-specific MOs were created, including cyclin B1 MO, cyclin B2 MO and tbp MO, in addition to cdk9 MO (Figure 4). To determine the MO specificity, the total RNA derived from 5 hpf embryos was examined, and a PAT assay was performed (Figure 4B and C). We tested a mixture of cyclin B1 MO plus cyclin B2 MO (Figure 4A and B). Zebrafish has the cyclin B orthologs B1 and B2, and their 3′-UTRs exhibit sequence similarity; indeed, there were 10-base homologies between cyclin B1 MO and cyclin B2 MO (Figure 4A). Cyclin B1 MO and cyclin B2 MO each affected its own target without oligonucleotides interference (Figure 4B). We also observed the specific action of MO when cdk9 MO and tbp MO were examined (Figure 4C). The effects of MO on the polyadenylation of mRNAs were confirmed by using RNA samples collected from 2 to 5 hpf embryos (Supplementary Figure S2A and S2B). In addition, each MO binds to a target 3′-UTR in a specific manner (Supplementary Figure S3). These results indicate that antisense MOs targeting the 3′-UTR of mRNAs affect polyadenylation in early zebrafish embryos in a specific manner.Figure 4.

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