The simian virus 40 late polyadenylation signal (SVLPA) is a complex and efficient polyadenylation signal which provides an ideal system for studying the mechanisms of polyadenylation, last exon definition and the coupling of polyadenylation and splicing during mRNA processing. In addition, studies of viral polyadenylation signals reveal how viruses manipulate cellular RNA processing mechanisms to their advantage. Understanding the structure/function relationships of viral polyadenylation signals provides the knowledge necessary to use polyadenylation as a specific target for anti-viral therapy. We have made significant progress in understanding the structure and function of the SVLPA signal. In particular, we have mapped functional secondary structure in its downstream region.
In Aim 1 we will expand the analyses of the primary and secondary structure of the SVLPA and other polyadenylation signals. This knowledge will allow: i) definition of specific and general features of polyadenylation signals; ii) studies of how polyadenylation factors utilize secondary structure; and iii) studies of the use of anti-sense oligonucleotides to target unique features of polyadenylation signals for gene specific inhibition of polyadenylation.
Aim 2 expands our studies to another viral system, the alternative polyadenylation of the major immediate early (MIE) gene of human cytomegalovirus (HCMV). The MIE gene's primary transcript is alternatively spliced and polyadenylated, forming several mRNAs. We have begun to analyze the structure and function of the two alternative polyadenylation signals and determine how their use may change during an infection. The set of studies proposed are the foundation for longer term goals to determine how alternative polyadenylation and splicing may be controlled during the course of a lytic infection. Significant progress has been made in understanding the coupling of polyadenylation and splicing. We have identified and purified an RNA-protein complex which correlates with coupling.
In Aim 3 we will continue to examine the factors in this coupling complex and study the mechanisms which underlie coupling. Using proteomic analyses we will identify the proteins in the coupling complex. These analyses will open new lines of research to characterize the functional connections between the factors in the complex and decipher the mechanisms which control processing at the level of coupling.
