Cloning And Functional Study Of RNA Binding Protein To An Upstream Regulatory Element (URE) Of Adenovirus

Adenovirus infection cycle in host cells is conveniently divided into early and late stages by the onset of DNA replication. Adenovirus has four early transcription units that are responsible for the synthesis of viral regulatory proteins, while the late transcription unit mainly encodes for viral structural proteins. The switch of early-to-late gene expression is strictly controlled by cell in all levels. The adenovirus major transcription unit (MLTU) has five transcription regions, L1 through L5, defined by its five poly A sites. The polyA site using efficiency in MLTU are different during adenovirus life cycle: the use of promoter proximal L1 poly A site is predominant over other poly A sites at early infection stage, which codes for non-structure proteins for viral encapsidation. However, all five polyA sites can be equally highly represented by adenovirus at late infection stage, which code for major viral structure proteins. Thus, the transition from early to late stage infection by adenovirus involves a change in mRNA expression from MLTU. The key question is how adenovirus can discriminatingly use different polyA sites in MLTU at different infection stage?It has been demonstrated that the MLTU contains an upstream repressor element (URE) at upstream of L1 poly A site. URE is revealed to influence steady state of L2 and L3 RNA transcript containing URE, and allow L1 to function as a dominant polyA site in a complex transcription unit at early infection stage. An in vitro UV cross linking experiment between URE RNA transcripts and HeLa nuclear extracts identified at least two potential nuclear factors with the molecular weights less than 30Kd can specifically bind to the URE. To further deepen our understanding of mechanism of URE's gene repression, we screened HeLa cDNA library with yeast three-hybrid method, then studied the function of a candidate RNA binding protein. We have found the candidate RNA binding protein play a part in URE's gene repression mechanism. It offers a new clue to further know the controlling mechanism of early-to late infection stage switch in adenovirus and the gene regulatory mechanism of genes at post-transcription level in nucleus.The yeast three-hybrid have three components: a protein fused with DNA binding domain, a hybrid RNA molecule, a protein fused with DNA activation domain. In a yeast three-hybrid system, hybrid RNA molecule is a "bait" to allure proper "prey" from a cDNA library.We firstly titered the library, and then amplified 3×10~7 independent clones of library, which is 10 fold of initial size of the library, to effectively maintain the size of amplified library. We get a large quantity of highly purified plasmid from amplified library. Next, after examining the RNA sequence of URE, we found URE contain consecutive four U bases, a...