Identification of a bidirectional splicing enhancer: differential involvement of SR proteins in 5' or 3' splice site activation.
The adenovirus E1A pre-mRNA undergoes alternative splicing whose modulation occurs during infection, through the use of three different 5' splice sites and of one major or one minor 3' splice site. Although this pre-mRNA has been extensively used as a model to compare the transactivation properties of SR proteins, no cis-acting element has been identified in the transcript sequence. Here we describe the identification and the characterization of a purine-rich splicing enhancer, located just upstream of the 12S 5' splice site, which is formed from two contiguous 9-nucleotide (nt) purine motifs (Pu1 and Pu2). We demonstrate that this sequence is a bidirectional splicing enhancer (BSE) in vivo and in vitro, because it activates both the downstream 12S 5' splice site through the Pu1 motif and the upstream 216-nt intervening sequence (IVS) 3' splice site through both motifs. UV cross-linking and immunoprecipitation experiments indicate that the BSE interacts with several SR proteins specifically, among them 9G8 and ASF/SF2, which bind preferentially to the Pu1 and Pu2 motifs, respectively. Interestingly, we show by in vitro complementation assays that SR proteins have distinct transactivatory properties. In particular, 9G8, but not ASF/SF2 or SC35, is able to strongly activate the recognition of the 12S 5' splice site in a BSE-dependent manner in wild-type E1A or in a heterologous context, whereas ASF/SF2 or SC35, but not 9G8, activates the upstream 216-nt IVS splicing. Thus, our results identify a novel exonic BSE and the SR proteins which are involved in its differential activity.
Bourgeois
C F
CF
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 67404 Illkirch C.U. de Strasbourg, France.
Popielarz
M
M
Hildwein
G
G
Stevenin
J
J
D017500:Adenovirus E1A Proteins / Q000235:genetics*; D001483:Base Sequence; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D029543:Nucleocytoplasmic Transport Proteins; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism*; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D000068103:Serine-Arginine Splicing Factors
D017500:Adenovirus E1A Proteins; D009687:Nuclear Proteins; D029543:Nucleocytoplasmic Transport Proteins; D010750:Phosphoproteins; D012322:RNA Precursors; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors