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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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The region 40-nt upstream from the zcdk9 3′-UTR end comprises the effective element for MO-mediated repression of zcdk9 mRNA. (A) Terminal sequence of the zcdk9 mRNA 3′-UTR and hybridization positions of antisense MOs. (B) Full repression of zcdk9 mRNA by injection of cdk9 MO, MO-5, MO-6 and MO-7. Embryos in which indicated MOs were injected (lanes 2 to 8) or uninjected (lane 1) were collected at 3 hpf. The total RNA was extracted, and a PAT assay was performed with PAT primers for cdk9 and tbp. (C) Extracts from embryos (5 hpf) in which indicated MOs were injected (lanes 2–8) or uninjected (lane 1) were subjected to 7.5% SDS–polyacrylamide gel electrophoresis. Blots were probed with anti-zCdk9, H-169 and anti-actin. HeLa cell NEs were also analyzed (lane 9) as a control.
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gks765-F2: The region 40-nt upstream from the zcdk9 3′-UTR end comprises the effective element for MO-mediated repression of zcdk9 mRNA. (A) Terminal sequence of the zcdk9 mRNA 3′-UTR and hybridization positions of antisense MOs. (B) Full repression of zcdk9 mRNA by injection of cdk9 MO, MO-5, MO-6 and MO-7. Embryos in which indicated MOs were injected (lanes 2 to 8) or uninjected (lane 1) were collected at 3 hpf. The total RNA was extracted, and a PAT assay was performed with PAT primers for cdk9 and tbp. (C) Extracts from embryos (5 hpf) in which indicated MOs were injected (lanes 2–8) or uninjected (lane 1) were subjected to 7.5% SDS–polyacrylamide gel electrophoresis. Blots were probed with anti-zCdk9, H-169 and anti-actin. HeLa cell NEs were also analyzed (lane 9) as a control.

Mentions: To address whether the 3′-UTR terminus plays an important role in MO-mediated repression, three antisense MOs were created, including MO-2 (−26 to −50), MO-3 (−51 to −75) and MO-4 (−76 to −100) (Supplementary Figure S1). The PAT assay and western blot analysis indicated that, of the five MOs listed in Supplementary Figure S1, only cdk9 MO exerted an inhibitory effect on both polyadenylation and translation. This finding suggests that the most terminal portion of the 3′-UTR is critical for inhibition. To verify this result, additional antisense MOs (Figure 2) were created, namely MO-5 (−6 to −30), MO-6 (−11 to −35) and MO-7 (−16 to 40), which showed inhibitory effects on both polyadenylation and translation that were greater than or similar to those of cdk9 MO. A modest effect on both polyadenylation and translation was apparent when MO-8 (−21 to −45) was examined (Figure 2B and C). These results indicate that the terminal 40 nt of the 3′-UTR act as the active region for MO-mediated inhibition.Figure 2.


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)

The region 40-nt upstream from the zcdk9 3′-UTR end comprises the effective element for MO-mediated repression of zcdk9 mRNA. (A) Terminal sequence of the zcdk9 mRNA 3′-UTR and hybridization positions of antisense MOs. (B) Full repression of zcdk9 mRNA by injection of cdk9 MO, MO-5, MO-6 and MO-7. Embryos in which indicated MOs were injected (lanes 2 to 8) or uninjected (lane 1) were collected at 3 hpf. The total RNA was extracted, and a PAT assay was performed with PAT primers for cdk9 and tbp. (C) Extracts from embryos (5 hpf) in which indicated MOs were injected (lanes 2–8) or uninjected (lane 1) were subjected to 7.5% SDS–polyacrylamide gel electrophoresis. Blots were probed with anti-zCdk9, H-169 and anti-actin. HeLa cell NEs were also analyzed (lane 9) as a control.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks765-F2: The region 40-nt upstream from the zcdk9 3′-UTR end comprises the effective element for MO-mediated repression of zcdk9 mRNA. (A) Terminal sequence of the zcdk9 mRNA 3′-UTR and hybridization positions of antisense MOs. (B) Full repression of zcdk9 mRNA by injection of cdk9 MO, MO-5, MO-6 and MO-7. Embryos in which indicated MOs were injected (lanes 2 to 8) or uninjected (lane 1) were collected at 3 hpf. The total RNA was extracted, and a PAT assay was performed with PAT primers for cdk9 and tbp. (C) Extracts from embryos (5 hpf) in which indicated MOs were injected (lanes 2–8) or uninjected (lane 1) were subjected to 7.5% SDS–polyacrylamide gel electrophoresis. Blots were probed with anti-zCdk9, H-169 and anti-actin. HeLa cell NEs were also analyzed (lane 9) as a control.
Mentions: To address whether the 3′-UTR terminus plays an important role in MO-mediated repression, three antisense MOs were created, including MO-2 (−26 to −50), MO-3 (−51 to −75) and MO-4 (−76 to −100) (Supplementary Figure S1). The PAT assay and western blot analysis indicated that, of the five MOs listed in Supplementary Figure S1, only cdk9 MO exerted an inhibitory effect on both polyadenylation and translation. This finding suggests that the most terminal portion of the 3′-UTR is critical for inhibition. To verify this result, additional antisense MOs (Figure 2) were created, namely MO-5 (−6 to −30), MO-6 (−11 to −35) and MO-7 (−16 to 40), which showed inhibitory effects on both polyadenylation and translation that were greater than or similar to those of cdk9 MO. A modest effect on both polyadenylation and translation was apparent when MO-8 (−21 to −45) was examined (Figure 2B and C). These results indicate that the terminal 40 nt of the 3′-UTR act as the active region for MO-mediated inhibition.Figure 2.

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