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

The polysomal pool of RNA contains uncapped 3′ mRNA cleavage fragments

<p><b>Copyright information:</b></p><p>Taken from "Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides"</p><p>Nucleic Acids Research 2005;33(1):114-125.</p><p>Published online 07 Jan 2005</p><p>PMCID:PMC546143.</p><p>© 2005, the authors © </p> () Strategy to examine the mRNA 5′ capping status by RNA ligation and subsequent RT–PCR. See Results for details. () Northern-blot analysis of RNA from cells cotransfected with DHBV and ASO DHBV795 followed by polysome analysis (top). Non-polysomal (N) and polysomal fractions (P) were pooled and the capping status of stable 3′ mRNA cleavage fragments was examined. Agarose gel photograph of the PCR products (bottom). Ligation and subsequent amplification of ∼0.4 kb PCR product indicated that the sample contained uncapped 3′ mRNA cleavage fragments. It should be noted that the two larger products were amplified from pgRNA and DHBV DNA due to internal binding of the primers. Amplification of these longer products did not reflect the capping status as it was independent of the ligation step. Total cellular RNA from DHBV-transfected cells without (negative control) and with ASO DHBV795 (positive control) was used as controls. () Six clones from the non-polysomal and polysomal pool each were cloned and sequenced. The position of the 5′ terminus of stable 3′ mRNA cleavage fragments corresponding to the RNase H cleavage site is indicated

Stable 3′ mRNA cleavage fragments are translated with moderate efficiency

<p><b>Copyright information:</b></p><p>Taken from "Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides"</p><p>Nucleic Acids Research 2005;33(1):114-125.</p><p>Published online 07 Jan 2005</p><p>PMCID:PMC546143.</p><p>© 2005, the authors © </p> () Translation of stable 3′ mRNA cleavage fragments was quantified using a preS luciferase fusion construct. The binding sites for different ASO and labeled probes are shown. The drawing is not to scale. () Northern-blot analysis with probes discriminating RNA cleavage fragments upon cotransfection of preS-Luc with different ASO. Faint bands consistent with 5′ cleavage fragments are indicated by arrows. () Immunoblotting of luciferase fusion proteins upon cotransfection with different ASO is depicted (top). Equal amounts of protein were loaded. Quantification of protein yield by luciferase activity (bottom). Values are given in percent compared to luciferase activity obtained after cotransfection of control ASO. The number of observations is given in brackets. Asterisks indicate a statistically significant difference compared with control ASO treated cells, whereas, the section mark symbol indicates a statistically significant difference between cells cotransfected with ASO DHBV795 and DHBV874

The DHBV preS region does not display IRES activity

<p><b>Copyright information:</b></p><p>Taken from "Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides"</p><p>Nucleic Acids Research 2005;33(1):114-125.</p><p>Published online 07 Jan 2005</p><p>PMCID:PMC546143.</p><p>© 2005, the authors © </p> () Schematic diagram of a dicistronic vector comprising EGFP and preS Luc is shown. () Immunoblotting of preS luciferase fusion proteins and EGFP upon cotransfection with ASO. Monocistronic preS-Luc served as positive control. Equal amounts of protein were loaded. Relative luciferase activity was compared to luciferase activity obtained after cotransfection of the monocistronic construct with control ASO and is given in percent. The number of observations is given in brackets

Purinergic P2Y₂ receptors promote neutrophil infiltration and hepatocyte death in mice with acute liver injury.

During progression of liver disease, inflammation affects survival of hepatocytes. Endogenous release of adenosine triphosphate (ATP) in the liver activates purinergic P2 receptors (P2R), which regulate inflammatory responses, but little is known about the roles of these processes in the development of acute hepatitis.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe