Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides.
Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) are used to suppress gene expression by inducing RNase H-mediated cleavage with subsequent degradation of the target mRNA. However, previous observations suggest that ASO/RNase H can also result in the generation of stable mRNA cleavage fragments and expression of truncated proteins. Here, we addressed the underlying translational mechanisms in more detail using hepadnavirus-transfected hepatoma cells as a model system of antisense therapy. Generation of stable mRNA cleavage fragments was restricted to the ASO/RNase H pathway and not observed upon cotransfection of isosequential small interfering RNA or RNase H-incompetent oligonucleotides. Furthermore, direct evidence for translation of mRNA fragments was established by polysome analysis. Polysome-associated RNA contained cleavage fragments devoid of a 5' cap structure indicating that translation was, at least in part, cap-independent. Further analysis of the uncapped cleavage fragments revealed that their 5' terminus and initiation codon were only separated by a few nucleotides suggesting a 5' end-dependent mode of translation, whereas internal initiation could be ruled out. However, the efficiency of translation was moderate compared to uncleaved mRNA and amounted to 13-24% depending on the ASO used. These findings provide a rationale for understanding the translation of mRNA fragments generated by ASO/RNase H mechanistically.
Hasselblatt
Peter
P
Department of Medicine II, University of Freiburg Hugstetter Strasse 55, D-79106 Freiburg, Germany. Hasselblatt@imp.univie.ac.at
Hockenjos
Birgit
B
Thoma
Christian
C
Blum
Hubert E
HE
Offensperger
Wolf-Bernhard
WB
D020121:5' Untranslated Regions; D000818:Animals; D002451:Cell Compartmentation; D045744:Cell Line, Tumor; D015613:Hepadnaviridae / Q000235:genetics* / Q000378:metabolism; D015611:Hepatitis B Virus, Duck / Q000235:genetics; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000494:pharmacology; D016914:Ribonuclease H / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology*
D020121:5' Untranslated Regions; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D013873:Thionucleotides; D016914:Ribonuclease H