Pre-mRNA splicing is one of the most important steps in control of gene expression. This essential step involves intron removal from a primary transcript and exon ligation to form a real message. In many cases, the mechanisms that regulate RNA splicing remain poorly understood. DNA tumor viruses such as cervical cancer-associated human papillomaviruses (HPV) and Kaposi's sarcoma-associated herpesvirus (KSHV) have several critical genes undergoing regulation by RNA splicing at post-transcriptional level. Our research focus is to understand the mechanisms that control viral RNA splicing and to look for new tools and molecular targets for antiviral and anticancer therapy at RNA level. Present studies in our laboratory focus on (1) identification of viral cis elements that are involved in regulation of the RNA splicing of viral structural and nonstructural genes including viral oncogenes in high and low risk HPVs and KSHV, (2) characterization of cellular splicing factors and viral proteins involved in processing of RNA splicing and (3) development of RNA interference as a new tool for antiviral and anticancer therapy. We have been utilizing HPV 16 and 18 E6 and E7 RNA transcripts as a first step to approach our goal. The E6 and E7 genes of HPV 16 and 18 are two major viral oncogenes and are expressed in almost every cancer cell of cervical carcinoma. E6 and E7 proteins inactivate cellular tumor suppressor proteins p53 and pRb, respectively, and play key roles in the induction of human cervical cancer. However, expression of E6 and E7 is complicated not only with their transcription as a bi-cistronic mRNA, but also with alternative splicing of their primary transcripts from which a large portion of E6 has been removed through using two alternative 3' splice sites within E6 coding regions. These alternatively spliced RNA species are termed E6*I and E6*II and form the majority of early viral transcripts both in cervical tumors and in tumor-derived cell lines. Ironically, transcripts for unspliced, full-length E6 are in extremely low abundance and sometimes it is hard to detect in many tumors or tumor-derived cell lines. We are analyzing HPV 16 and 18 E6-E7 RNA splicing patterns in cervical cancer-derived cell lines with various strategies and have successfully established an in vitro splicing system to identify the viral cis element that controls the alternative splicing of the E6-E7 pre-mRNAs. Works are in progress on the characterization of these elements and their interaction with splicing factors and viral proteins. Another approach in our laboratory is to determine how cellular splicing factors are involved in viral RNA splicing. We are using a bovine papillomavirus type 1 (BPV-1, a prototype virus in papillomavirus family) late pre-mRNA as our model to address this question since this pre-mRNA has two alternative 3' splice sites (3'ss). Switching from one 3' ss to another in splicing of this transcript relates to keratinocyte differentiation and involves viral cis elements interacting with cellular splicing factors. We have established a series of cell lines with stable transfection of BPV-1 late genes. These cell lines have a cellular splicing factor ASF/SF2 under the control of a tetracycline (tet)-repressible promoter. Using these cell lines, we have demonstrated that the splicing factor ASF/SF2 is required for such a switch of 3' ss usage, but not essential. Activation of the cells doesn't recover tet-repressed ASF/SF2 expression, but restores the default pattern of 3' ss utilization though activation of other splicing factors. Currently, we are focusing on the characterization of transcription and polyadenylation coupling with this feature of the splicing. Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus (HHV8) is a newly identified human gamma herpesvirus strongly associated with development of KS, body cavity-based B-cell lymphoma and Castleman's disease. Currently, our lab is focusing on the KSHV K8 and K8.1 which are two juxtaposed, but unrelated genes posited from nt 74850 to nt 76730 of the virus genome. However, both genes share a single poly (A) site at nt 76714. The K8 gene consists of 4 exons and 3 introns which are alternatively spliced during the viral gene expression. The K8.1, although sharing the exon 4 with K8, utilizes the intron 3 of K8 as its own coding region which is also alternatively spliced to the exon 4. We have extensively profiled the RNA splicing patterns of the K8 and K8.1 in KSHV+ JSC-1 cells and identified presence of a bicistronic pre-mRNA which has three alternative 5' splice site in its intron 3. Further experiments show that this bicistronic RNA could be used as a precursor of both K8 and K8.1 mRNAs. Our current focus is (1) to understand how three 5' splice sites in this bicistronic RNA are alternatively selected during the viral life cycle and B cell differentiation and (2) to identify viral cis elements and trans-acting factors involved in this regulation. ---AID related (50%)
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