Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation.
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.
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
Tadashi
T
Wada
Masatoshi
M
Hara
Takuya
T
Taneda
Cao
C
Qingfu
Ryouhei
R
Takata
Kanako
K
Moro
Kei
K
Takeda
Takeo
T
Kishimoto
Hiroshi
H
Handa
D020413:3' Untranslated Regions; D000818:Animals; D047288:Asterina / Q000235:genetics / Q000378:metabolism; D019926:Cyclin B / Q000235:genetics / Q000378:metabolism; D042863:Cyclin-Dependent Kinase 9 / Q000037:antagonists & inhibitors / Q000235:genetics; D015536:Down-Regulation; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D007267:Injections; D060172:Morpholinos / Q000008:administration & dosage / Q000494:pharmacology*; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage / Q000494:pharmacology*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011061:Poly A / Q000378:metabolism*; D026723:Polyadenylation / Q000187:drug effects; D014176:Protein Biosynthesis / Q000187:drug effects*; D023121:RNA, Messenger, Stored / Q000737:chemistry / Q000378:metabolism*; D015027:Zebrafish / Q000196:embryology / Q000235:genetics / Q000378:metabolism; D029961:Zebrafish Proteins / Q000037:antagonists & inhibitors / Q000235:genetics
D020413:3' Untranslated Regions; D019926:Cyclin B; D060172:Morpholinos; D016376:Oligonucleotides, Antisense; D023121:RNA, Messenger, Stored; D029961:Zebrafish Proteins; D011061:Poly A; C517155:Cdk9 protein, zebrafish; D042863:Cyclin-Dependent Kinase 9