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Sequences involved in the control of adenovirus L1 alternative RNA splicing
J P Kreivi1, K Zerivitz, G Akusjärvi
1Department of Microbial Genetics, Karolinska Institute, Stockholm, Sweden.
Abstract:
During an adenovirus infection the expression of mRNA from late region L1 is temporally regulated at the level of alternative 3' splice site selection to produce two major mRNAs encoding the 52,55K and IIIa polypeptides. The proximal 3' splice site (52,55K) is used at all times of the infectious cycle whereas the distal site (IIIa) is used exclusively late after infection. We show that a single A branch nucleotide located at position -23 is used in 52,55K splicing and that two A's located at positions -21 and -22 are used in IIIa splicing. Both 3' splice sites were active in vitro in nuclear extracts prepared from uninfected HeLa cells. However, the efficiency of IIIa splicing was only approximately 10% of 52,55K splicing. This difference in splice site activity correlated with a reduced affinity of the IIIa, relative to the 52,55K, 3' splice site for polypyrimidine tract binding proteins. Reversing the order of 3' splice sites on a tandem pre-mRNA resulted in an almost exclusive IIIa splicing indicating that the order of 3' splice site presentation is important for the outcome of alternative L1 splicing. Based on our results we suggest a cis competition model where the two 3' splice sites compete for a common RNA splicing factor(s). This may represent an important mechanism by which L1 alternative splicing is regulated.
Insights
Adenovirus L1 alternative splicing temporally regulates mRNA production using distinct branch nucleotides. A cis competition model suggests splice sites compete for factors, impacting 52,55K and IIIa polypeptide expression.
Area of Science:
- Molecular Biology
- Virology
- RNA Splicing
Background:
- Adenovirus infection involves temporal regulation of late region L1 mRNA expression.
- Alternative 3' splice site selection produces mRNAs for 52,55K and IIIa polypeptides.
- The proximal 52,55K splice site is constitutively used, while the distal IIIa site is used late in infection.
Purpose of the Study:
- To investigate the molecular mechanisms regulating alternative 3' splice site selection in adenovirus L1 splicing.
- To identify the specific branch nucleotides involved in 52,55K and IIIa splicing.
- To elucidate the role of splice site affinity for RNA-binding proteins and splice site order.
Main Methods:
- In vitro splicing assays using nuclear extracts from HeLa cells.
- Analysis of branch nucleotide usage for proximal (52,55K) and distal (IIIa) splice sites.
- Assessment of polypyrimidine tract binding protein affinity for different splice sites.
- Experimentation with tandem pre-mRNAs to evaluate splice site order effects.
Main Results:
- A single adenine at -23 is used for 52,55K splicing; two adenines at -21 and -22 are used for IIIa splicing.
- In vitro, IIIa splicing efficiency was approximately 10% of 52,55K splicing.
- Reduced affinity of the IIIa splice site for polypyrimidine tract binding proteins was observed.
- Reversing splice site order on tandem pre-mRNAs led to almost exclusive IIIa splicing.
Conclusions:
- Splice site selection is influenced by specific branch nucleotide usage and affinity for RNA-binding proteins.
- The order of 3' splice site presentation is critical for determining the outcome of alternative L1 splicing.
- A cis competition model is proposed, where splice sites compete for common splicing factors, regulating adenovirus L1 alternative splicing.