The nuclear-replicating DNA viruses provide an excellent model systems for the study of polyadenylation (PA) since pre-mRNAs transcribed from their genes utilize cellular PA mechanisms. However, the rationale for studying PA in viral systems is far more compelling. Due to the their special needs the DNA viruses appear to call on the cell's full repertoire of PA mechanisms to define and polyadenylate their mRNAs. These viral PA signals must often deal with last exons much larger than those of most cellular pre-mRNAs. In addition, many viral PA signals must function very efficiently to process the large amounts of RNA which may be produced from viral genes during infection. Hence the viruses appear to provide accentuated PA systems from which we can examine not only PA mechanisms but also how viruses manipulate them. We have characterized the SV40 and HIV PA signals and found that they may be more complex than the majority of their cellular counterparts. In addition to the AAUAAA and downstream U- or GU-rich elements found in most PA signals, many viral signals also contain sequences upstream of the AAUAAA, known as upstream elements (USEs), which affect the efficiency of PA. We have found that a component of the splicing apparatus, the U1snRNP-A protein (U1-A) interacts with the upstream element of the SV4O late PA signal. This indicates a mechanism by which interactions between the splicing and PA complexes are facilitated through binding the snRNP to the pre-pre-mRNA close to the AAUAAA. Our data suggest that one such interaction is between the U1-A protein and the 160 kD component of the cleavage and PA specificity factor (CPSF), a major component of the PA complex. We suggest that this represents communication between the splicing and PA complexes resulting in definition of the last exon of the mRNA, promotion of the removal of the last intron and increased efficiency of PA. To further our studies and test our model we propose to: 1) Determine the utilization of the USEs and other PA signal elements in the coupling of PA and the removal of the last intron. 2) Continue our studies of the function of the U1-A protein, as well as other U1 snRNP proteins, non-snRNP splicing factors and hnRNPs in PA and last exon definition. Our emphasis will be directed toward the nature of interactions of specific proteins with other proteins and RNA. 3) Further define viral PA signals. We will continue to examine the PA signals of SV4O and HIV, as well as other viral and cellular genes, to define the nature of USEs and determine their characteristics. We will also perform a detailed definition of the SV40 downstream elements and their utilization. 4) Identify alternate forms of U1-A protein and other proteins which interact with U1-A protein.
