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

Hara, Masatoshi; Taneda, Takuya; Cao, Qi; Takata, Ryouhei; Moro, Kanako; Takeda, Kei; Kishimoto, Takeo; Handa, Hiroshi

doi:10.1093/nar/gks765

Cited by 18 publications

(15 citation statements)

References 20 publications

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“…Synthesis of cDNA with biotinylated 3 ′ adaptor ligation was performed using small RNA Cloning Kit (Takara Bio) as reported previously (Wada et al 2012), followed by PCR using a gene-specific primer and a 3 ′ adaptor primer (primer sets are listed below). In some experiments, in order to increase the amount of DNA available for cloning, secondary nested PCR was performed using internal (nested) PCR primers (primer sets for the second amplification are listed below).…”

Section: Rt-pcr Withmentioning

confidence: 99%

“…To create pcDNAsfcycB3 ′ UTR1, the 3 ′ UTR of starfish cyclin B cDNA was amplified by RT-PCR using a sfcycB Fwd primer 1 and a (dT)12-anchor primer (5 ′ -GCGAGCTCCGCGGCCGCG TTTTTTTTTTTT-3 ′ ) (Wada et al 2012), which was TA-cloned into vector pCR2.1-TOPO (Invitrogen).…”

Section: Dna Cloning and Plasmids For In Vitro Rna Synthesismentioning

confidence: 99%

“…Therefore, the actual 3 ′ terminal sequences of cyclin B mRNAs of oocytes remain unknown in many animals. In this study, we applied adaptor ligation to 3 ′ termini of mRNAs (Wada et al 2012) in starfish oocytes to determine whether short poly(A) tails of cyclin B mRNAs are modified.…”

Section: Introductionmentioning

confidence: 99%

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Hormonal stimulation of starfish oocytes induces partial degradation of the 3′ termini of cyclin B mRNAs with oligo(U) tails, followed by poly(A) elongation

Ochi

Chiba

2016

RNA

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In yeast, plant, and mammalian somatic cells, short poly(A) tails on mRNAs are subject to uridylation, which mediates mRNA decay. Although mRNA uridylation has never been reported in animal oocytes, maternal mRNAs with short poly(A) tails are believed to be translationally repressed. In this study, we found that 96% of cyclin B mRNAs with short poly(A) tails were uridylated in starfish oocytes. Hormonal stimulation induced poly(A) elongation of cyclin B mRNA, and 62% of long adenine repeats did not contain uridine residues. To determine whether uridylated short poly(A) tails destabilize cyclin B mRNA, we developed a method for producing RNAs with the strict 3 ′ terminal sequences of cyclin B, with or without oligo(U) tails. When we injected these synthetic RNAs into starfish oocytes prior to hormonal stimulation, we found that uridylated RNAs were as stable as nonuridylated RNAs. Following hormonal stimulation, the 3 ′ termini of short poly(A) tails of synthesized RNAs containing oligo(U) tails were trimmed, and their poly(A) tails were subsequently elongated. These results indicate that uridylation of short poly(A) tails in cyclin B mRNA of starfish oocytes does not mediate mRNA decay; instead, hormonal stimulation induces partial degradation of uridylated short poly(A) tails in the 3 ′ -5 ′ direction, followed by poly(A) elongation. Oligo(U) tails may be involved in translational inactivation of mRNAs.

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Section: Rt-pcr Withmentioning

confidence: 99%

Section: Dna Cloning and Plasmids For In Vitro Rna Synthesismentioning

confidence: 99%

See 1 more Smart Citation

Hormonal stimulation of starfish oocytes induces partial degradation of the 3′ termini of cyclin B mRNAs with oligo(U) tails, followed by poly(A) elongation

Ochi

Chiba

2016

RNA

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“…Morpholinos have also been shown to block intronic splice silencers (Bruno et al, 2004) and exonic splice enhancers (McClorey et al, 2006), redirecting splicing. Morpholinos binding poly(A) signal sequences can inhibit poly(A) tailing (Wada et al, 2012). Morpholinos binding translocation sequences can inhibit directed movement of RNA within cells (Arthur et al, 2009).…”

Section: Background Informationmentioning

confidence: 99%

Using Morpholinos to Control Gene Expression

Moulton

2017

CP Nucleic Acid Chemistry

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Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis-nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos. © 2017 by John Wiley & Sons, Inc.

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“…They efficiently inhibit gene expression by hampering pre‐mRNA splicing or blocking translation . The latter is achieved by designing PMOs that either bind to complimentary base pairs in the 5′ UTR (leader sequence), the first 25 bases 3′ to the AUG translational start site or base pairs in the 3′‐UTR . Their extraordinary stability, backbone neutrality, water solubility and predictive antisense properties make them promising therapeutics for cancer and other genetic diseases.…”

Section: Introductionmentioning

confidence: 99%

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

et al. 2017

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Cationic guanidinium and phosphonium functionalized cytosine morpholino tetramer (G3) and trimer (P3) have been reported for the first time. They show efficient cellular uptake properties in full growth medium, quantified by fluorescence activated cell sorting (FACS) and visualized by fluorescence microscopy. An interesting feature of these transporters is their localization in the nucleolus or the rest of nucleus, determined by the nature of cationic moieties attached with cytosine base. Co-staining with MitoTracker Deep Red depicts their ability to penetrate mitochondrial membrane. Fluorescence imaging displays their uniform distribution throughout zebrafish larvae with no significant toxicity. FACS analysis at 4 8 C indicated that transfection of G3 transporter is energy dependent whereas P3 is energy independent. When covalently conjugated, G3 is capable of transporting a 25-mer phosphorodiamidate morpholino oligomer (PMO) into the cells. It exhibits antisense effect against Gli1 gene in hedgehog pathway, confirmed in preliminary b-glactosidase assay performed in Ptch1 -/cells. Further antisense effect was confirmed when G3-conjugated zebrafish Gli1-PMO was injected into embryo to obtain Gli1dependent phenotypes. To the best of our knowledge, herein, we first report that molecular transporters with a minimum number of three cationic groups are capable of efficiently passing through plasma, nuclear and mitochondrial membranes and show distribution in zebrafish larvae.[a] Dr. Figure 1. Chemical structures of (a) DNA, (b) PMO, transporters (c) G3 (1) and (d) P3 (2).

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

Cited by 18 publications

References 20 publications

Hormonal stimulation of starfish oocytes induces partial degradation of the 3′ termini of cyclin B mRNAs with oligo(U) tails, followed by poly(A) elongation

Hormonal stimulation of starfish oocytes induces partial degradation of the 3′ termini of cyclin B mRNAs with oligo(U) tails, followed by poly(A) elongation

Using Morpholinos to Control Gene Expression

Cationic Cytosine Morpholino‐Based Transporters: Synthesis and Regulation of Intracellular Localization

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