| Summary: | 碩士 === 國立陽明大學 === 微生物暨免疫學研究所 === 90 === Hepatitis B virus (HBV) is a small DNA virus. Multiple RNAs, including the 3.5- kb genomic RNA and 2.4-kb, 2.1-kb, 0.8-kb sub-genomic RNAs, are produced. These transcripts originate from different promoters, but utilize the same polyadenylaton site. In addition, multiple spliced RNAs generated from the 3.5-kb RNA have been identified in both HBV-infected human livers and the HBV transient expression system. Among them, the 2.2-kb singly-spliced species is the most abundant one. In contrast to cellular mRNAs, the introns of which are rapidly spliced out, both the HBV 3.5-kb RNA and the spliced RNAs can be maintained stably in cytoplasm. Similar phenomenon is obser- ved in retroviruses. The HBV 3.5-kb RNA serves as templates for both viral DNA repli- cation and protein synthesis, a control mechanism must exist to maintain the appropriate level of the 3.5-kb RNA.
To study such a control mechanism, splicing efficiency was analyzed by S1 nucle- ase mapping using RNA isolated from a HBV transient transfection system. We first examined whether HBV splicing is regulated by the suboptimal splicing signal. Indeed, when the major 5’ splice donor site on the HBV expression plasmid was changed to the consensus sequence, the splicing efficiency increased dramatically. On the other hand, studies in retrovirus suggest a role of exon sequences in maintenance of balanced splic- ing in the context of the suboptimal signals. Analysis of the sequence of the HBV termi- nal exon adjacent to the 5’ splice donor site revealed the existence of sequences homo- logous, respectively, to exon splicing silencer (ESS) and exon splicing enhancer (ESE) of the HIV tat exon. When the putative HBV ESS sequence was deleted, the splicing efficiency increased to the level similar to that observed in cells transfected with const- ruct of optimal splice donor site, suggesting this element acts as a splicing silencer. Fur- thermore, our study showed that ESS is unable to suppress splicing of RNA with an opt- imized splice donor site. On the other hand, deletion of the putative ESE element has no effect on RNA splicing. However, when both ESS and ESE were deleted, the splicing efficiency returned to the level observed in the wild-type construct, suggesting the ESE counterbalances the effect of the ESS.
To study trans-cellular factor(s) that involving in HBV splicing regulation, we tried to raise in vivo level of SR proteins or hnRNP A1 protein via expression of cDNAs cod- ing for individual members and studied the effect of such manipulation on HBV splicing efficiency. Our preliminary results show the over-expression of hnRNP A1 enhances the production of 2.2-kb singly-spliced RNA.
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