Figure 4.

The polysomal pool of RNA contains uncapped 3′ mRNA cleavage fragments. (A) Strategy to examine the mRNA 5′ capping status by RNA ligation and subsequent RT–PCR. See Results for details. (B) 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. (C) 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.