Target-specific arrest of mRNA translation by antisense 2'-O-alkyloligoribonucleotides.
We describe a novel experimental approach to investigate mRNA translation. Antisense 2'-O-allyl oligoribonucleotides (oligos) efficiently arrest translation of targeted mRNAs in rabbit reticulocyte lysate and wheat germ extract while displaying minimal non-specific effects on translation. Oligo/mRNA-hybrids positioned anywhere within the 5' UTR or the first approximately 20 nucleotides of the open reading frame block cap-dependent translation initiation with high specificity. The thermodynamic stability of hybrids between 2'-O-alkyl oligos and RNA permits translational inhibition with oligos as short as 10 nucleotides. This inhibition is independent of RNase H cleavage or modifications which render the mRNA untranslatable. We show that 2'-O-alkyl oligos can also be employed to interfere with cap-independent internal initiation of translation and to arrest translation elongation. The latter is accomplished by UV-crosslinking of psoralen-tagged 2'-O-methyloligoribonucleotides to the mRNA within the open reading frame. The utility of 2'-O-alkyloligoribonucleotides to arrest translation from defined positions within an mRNA provides new approaches to investigate mRNA translation.
Johansson
H E
HE
Gene Expression Programme, European Molecular Biology Laboratory, Heidelberg, Germany.
Belsham
G J
GJ
Sproat
B S
BS
Hentze
M W
MW
D001483:Base Sequence; D002474:Cell-Free System; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D005363:Ficusin; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D011388:Prolactin / Q000096:biosynthesis; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis; D016372:RNA, Antisense / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000096:biosynthesis; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000096:biosynthesis
D009843:Oligoribonucleotides; D011498:Protein Precursors; D016372:RNA, Antisense; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; C017671:preprolactin; D011388:Prolactin; D015500:Chloramphenicol O-Acetyltransferase; D005363:Ficusin