The objective of this proposal is to characterize the processing mechanisms of messenger RNA synthesized by adenovirus type 2 during productive infection. These studies will focus on viral and cellular mRNA splicing. We have developed an in vitro splicing system that utilizes as substrates adenovirus 2 pre-RNAs synthesized in vitro from plasmid DNAs containing the Salmonella Phage 6 promoter. Several Kinetic parameters as well as the requirements for the in vitro splicing reaction were defined in this system. Recently, we have obtained a 108-fold purification of the splicing activity from crude nuclear extracts. In this application we propose to further purify and identify the individual components of mRNA splicing. We will attempt to identify potential RNA splicing intermediates with the different purified fractions. At the same time we will attempt to identify splicing intermediates in in vivo productively Ad2 infected cells. Studies will be carried out to determine the precise role and mechanism of the U1 snRNPs in in vitro mRNA splicing. We will analyze the nucleotide sequence requirement for E2 pre-RNA in vitro splicing, and also the ribonucleoprotein (RNPs) formation of the pre-RNAs in the presence of purified fractions. In addition, mRNA splicing of several viral genes will be studied in vitro. We will attempt first to reproduce in vitro the observed splicing patterns of several viral genes and second to purify and identify the factor(s) involved in the control of processing of viral mRNAs.
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