A Structured RNA in Hepatitis B Virus Post-transcriptional Regulatory Element Represses Alternative Splicing in a Sequence-independent and Position-dependent Manner

Hepatitis B virus (HBV) transcripts are subjected to multiple splicing decisions, but the mechanism of splicing regulation remains poorly understood. In this study, we used a well-investigated alternative splicing reporter to dissect splicing regulatory elements residing in the post-transcriptional regulatory element (PRE) of HBV. A strong intronic splicing silencer (ISS) with a minimal functional element of 105 nucleotides (referred to as PRE-ISS) was identified and, interestingly, both the sense and antisense strands of the element were found to strongly suppress alternative splicing in multiple human cell lines. PRE-ISS folds into a double-hairpin structure, in which substitution mutations disrupting the double-hairpin structure abolish the splicing silencer activity. Although it harbors two previously identified binding sites for polypyrimidine tract binding protein, PRE-ISS represses splicing independent of this protein. The silencing function of PRE-ISS exhibited a strong position dependence, decreasing with the distance from affected splice sites. PRE-ISS does not belong to the intronic region of any HBV splicing variants identified thus far, preventing the testing of this intronic silencer function in the regulation of HBV splicing. These findings, together with the identification of multiple sense-antisense ISSs in the HBV genome, support the hypothesis that a sequence-independent and structure-dependent regulatory mechanism may have evolved to repress cryptic splice sites in HBV transcripts, thereby preventing their aberrant splicing during viral replication in the host.