Regulation of mRNA synthesis in mammalian cells occurs at multiple stages, transcription, polyadenylation, RNA splicing and transport. At a mechanistic level, little is known about the process of splicing of mRNA precursors and components that effect the efficiency of RNA processing and transport. Understanding these processes at a molecular level would make the investigation of their role in complex biological phenomena such as differentiation, development and oncogenesis possible. We have recently shown that a purified RNA precursor containing the first and second exons of the major transcription unit of adenovirus can be accurately spliced in a soluble extract of mammalian cells. This reaction probably requires the presence of small ribonucleoprotein particles as it is inhibited by addition of antiserum that recognizes U1 RNP. We propose to further elucidate the biochemical mechanism of splicing of mRNA precursors using this system. The study will precede by determination of the structure of intermediates in the splicing process and the excised intervening sequence. In addition, the specific sequences required for splicing in vitro will be probed by synthesis of substrate RNA from variant templates. The role of snRNP in splicing of mRNA precursors will be investigated by fractionation of components in the extract required for synthesis of specific intermediates. Ultimately, we hope to reconstruct the reaction with purified factors. RNA processing and transport are critical steps in the synthesis of mRNAs from the major late transcriptional unit of adenovirus. We will study these processes by making adenovirus recombinants containing a short late transcription unit in the E1 region of Ad5. This transcription unit will contain the major late promoter and sequences for the tripartite leader in a cDNA conformation. The third leader will end in its normal 5' SS. We will manipulate this short late transcription unit to determine the sequence signals important in regulation of late mRNA synthesis, transport, and translation.
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