title: The second RNA-binding domain of the human splicing factor ASF/SF2 is the critical domain controlling adenovirus E1A alternative 5'-splice site selection.
abstract: The human splicing factor ASF/SF2 (alternative splicing factor/splicing factor 2) is modular in structure with two RNA-binding domains (RBD1 and RBD2) and a C-terminal domain rich in arginine-serine dipeptide repeats. ASF/SF2 is an essential splicing factor that also functions as an important regulator of alternative splicing. In adenovirus E1A (early region 1A) alternative pre-mRNA splicing, ASF/SF2 functions as a strong inducer of proximal 5'-splice-site selection, both in vitro and in vivo. In the present study, we tested the functional role of individual domains of ASF/SF2 in alternative splicing in vitro. We show that ASF/SF2-RBD2 is the critical domain controlling E1A alternative splicing. In fact, RBD2 alone is sufficient to mimic the activity of the full-length ASF/SF2 protein as an inducer of proximal 5'-splice-site selection in vitro. The RBD2 domain induces a switch to E1A-proximal 5'-splice-site usage by repressing distal 12 S splicing and simultaneously stimulates proximal 13 S splicing. In contrast, the ASF/SF2-RBD1 domain has a more general splicing enhancer phenotype and appears to stimulate preferentially cap-proximal 5'-splice-site selection. Furthermore, the SWQDLKD motif, which is conserved in all SR proteins (serine/arginine-rich proteins) containing two RBDs, and the ribonucleoprotein-1-type RNA recognition motif were both found to be necessary for the alternative splice-site-switching activity of ASF/SF2. The RNP-1 motif was necessary for efficient RNA binding, whereas the SWQDLKD motif most probably contributes by functioning as a surface-mediating critical protein-protein contact during spliceosome assembly.
authors.affiliation: Department of Medical Biochemistry and Microbiology, Uppsala University, BMC, Box 582, 751 23 Uppsala, Sweden.
authors.forename: Vita
authors.initials: V
authors.lastname: Dauksaite
authors.forename: Göran
authors.initials: G
authors.lastname: Akusjärvi
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D017500:Adenovirus E1A Proteins / Q000378:metabolism; D017398:Alternative Splicing / Q000235:genetics / Q000502:physiology; D020816:Amino Acid Motifs / Q000235:genetics / Q000502:physiology; D000595:Amino Acid Sequence / Q000502:physiology; D036022:Capsid Proteins / Q000502:physiology; D017124:Conserved Sequence / Q000235:genetics / Q000502:physiology; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D006367:HeLa Cells / Q000737:chemistry / Q000378:metabolism / Q000821:virology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics / Q000502:physiology; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D010455:Peptides / Q000235:genetics / Q000502:physiology*; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D012322:RNA Precursors / Q000378:metabolism; D022821:RNA Splice Sites / Q000502:physiology; D012326:RNA Splicing / Q000502:physiology; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012097:Repressor Proteins / Q000502:physiology; D000068103:Serine-Arginine Splicing Factors
chemical_list: D017500:Adenovirus E1A Proteins; D036022:Capsid Proteins; D009687:Nuclear Proteins; D010455:Peptides; D012322:RNA Precursors; D022821:RNA Splice Sites; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D000068103:Serine-Arginine Splicing Factors