Studies Of The HBV Posttranscriptional Regulatory Element Structure And Its Function In MRNA Splicing

HBV posttranscriptional regulatory element is an important cis-acting element within all HBV RNA unspliced transcript. Its most crucial function identified to date is that it facilitates the nucleocytoplasmic transport of the unspliced preS/S transcript, which encodes the HBV S proteins. Recently it was discovered that PRE is an exonic splicing enhancer (ESE) in HBV pgRNA splicing. However, we discovered that PRE might also be a potential splicing silencer, whose function may be dependent on its position in context and the local secondary structure formed by itself.We first identified PRE as a strong intronic splicing silencer (ISS) in a reporter plasmid, pZW8-SMN1, which was developed earlier in our lab. The full sequence of PRE was dismantled to pieces of varied lengths, and a gradual length restoration suggested that the minimal functional element within the full length PRE is a 105-nt fragment within its 3'end, which was named pre2bcd hereinafter. Further experiments showed that this fragment has a strong position dependency, and its silencing effect peaks at 82nt 5'towards the 3'splice site of exon 7. We found that in ealier reports polypyrimidine tract binding protein (PTB) was reported to bind PRE through two nearly consecutive binding sites, and that pre2bcd possesses both of them. Later tests showed that neither loss of both PTB binding sites nor the upregulation and downregulation of PTB itself has remarkable effect on pre2bcd's silencing function in the reporter plasmid. We also found that pre2bcd has little effect upon HBV pgRNA splicing, which is not unexpected considering its postion dependent manner.. Lastly Rnase footprinting assay was performed to probe the secondary structure of this fragment, which leads to the discovery that pre2bcd is a somewhat ordered structure featuring two stem-loop motifs that were later named SL1 and SL2. Our results indicate a possibility that pre2bcd's function is dependent on its own local structure and position in context rather than the common mechanisms of splicing regulatory elements which directly recruit trans-acting factors to their binding sites.Our results indicate that HBV PRE harbors an alternative splicing inhibitory element, which functions through inhibiting nearby alternative splice sites. In HBV genome there are several predicted 3'splice sites (AG) within PRE region. Our results suggest a possible splicing regulatory mechanism in HBV where distal splice site choices are promoted by the inhibition of proximal splice sites.