Abstract
To identify new sequence elements in the promoter that affect splicing patterns of pre-mRNAs, we analyzed effects of different promoters on alternative splicing of model reporter genes. We compared the E1a alternative splicing pattern in transcripts expressed from the full-length cytomegalovirus, SV40 early, or a hybrid cytomegalovirus/SV40 early promoter and found that the hybrid promoter improved selection of the suboptimal E1a 5'SS-1. Expressing RNA from the hybrid promoter also enhanced selection of suboptimal splice sites in other alternatively spliced reporter genes, demonstrating the generality of this effect. Unlike previously defined promoter elements shown to affect alternative splicing, which were located in the enhancer/upstream activating sequences, the motif identified in this work is positioned within the core promoter; it is comprised of eight T-residues directly upstream of the SV40 early TATA box. This motif was previously implicated in DNA bending and negative regulation of transcription. Together, these results suggest that the identity of transcription complex assembled in the core promoter-dependent fashion can affect splice site selection during pre-mRNA splicing, perhaps by influencing the processivity of transcription elongation.
Highlights
Eukaryotic promoters typically contain a core segment that recruits general transcription factors (GTFs) and an upstream activating sequence (UAS) that provides binding sites for transcriptional activators [7]
Transcription initiation, promoter clearance, and elongation are all affected by the PAF complex, which interacts with both initiation and elongation factors and affects 3Ј end processing and mRNA surveillance; this is consistent with a role for PAF in modulating multiple aspects of co-transcriptional events during mRNA biogenesis [17]
E1a transcripts initiated from a hybrid CMV/SV40 early (C/S) promoter exhibited altered splicing patterns as compared with transcripts initiated from full-length CMV or SV40 early promoters
Summary
Eukaryotic promoters typically contain a core segment that recruits general transcription factors (GTFs) and an upstream activating sequence (UAS) that provides binding sites for transcriptional activators [7]. Mutations within the T-stretch element directly upstream of the TATA box in SV40 core promoter change these alternative splicing patterns, identifying this sequence motif as responsible for the observed promoter effect.
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