The simian virus 40 late polyadenylation (SVLPA) signal is an ideal system to study not only the mechanisms of polyadenylation (PA) but also the means by which a virus can manipulate these mechanisms for its advantage. The SVLPA signal is both structurally more complex and functionally more efficient than most cellular PA signals, allowing it to take full advantage of cellular PA mechanisms. We have made significant progress in understanding the structure-function relationship of the SVLPA signal. In particular, we have determined that the defined elements of the signal form functionally important secondary and tertiary RNA structures which are important for: 1) the efficiency of PA; 2) the coupling of PA and splicing; and 3) last exon definition. Additional experiments suggest that the ability to form specific higher-order structures may be required for efficient utilization of viral and cellular PA signals in general. Further characterization of the structure-function relationship of virus PA signals will continue to give insight into PA mechanisms and will indicate how viral PA signals may be used as specific targets for anti-viral therapy. To these ends we propose four aims.
Aim 1 : We will continue to determine the secondary and tertiary structure of the SVLPA. To establish higher-order RNA structure as a more general feature of PA2 signals, we will analyze the basic structure of other viral and cellular PA signals, e.g. SV40 early, HIV, globin and synthetic PA signals, as well as the two polyadenylation signals of the major immediate early gene of human cytomegalovirus.
Aim 2 : We will continue to analyze the mechanisms of last exon definition and the coupling of PA and splicing using an RNA substrate containing the SVLPA with an upstream splicing cassette. We are analyzing the effects of mutating individual elements of splice sites and PA signals, such as those identified in Aim 1, on PA and coupling.
Aim 3 : We will continue our studies of factors involved in coupling of splicing and PA. Initially this will be a continuation of our analysis of the snRNP-free U1A protein complexes (SF-A complexes) which are involved in both splicing and PA. Identification of the proteins in the complexes will aid in determining how the complexes are involved with other splicing and PA factors to mediate coupling. The data from Aim 2 will also indicate factors potentially involved in coupling.
Aim 4 : We will determine whether specific disruption of the folded structure of a PA signal by anti-sense oligonucleotides specifically inhibits both PA and coupling, potentially providing a specific anti-viral therapeutic technique.
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