Related Experiment Videos
Splice site selection in polyomavirus late pre-mRNA processing
D B Batt1, L M Rapp, G G Carmichael
1Department of Microbiology, University of Connecticut Health Center, Farmington 06030.
Abstract:
Polyomavirus late pre-mRNAs contain one 5' splice site and two message body 3' splice sites, which are not used at equal frequencies. As a result of alternative splicing, the total late mRNA population consists of about 5% mVP2 (no message body splice chosen), about 15% mVP3 (promoter-proximal 3' splice site chosen), and about 80% mVP1 (promoter-distal 3' splice site chosen). To determine whether it is splice site strength that determines the ratio of spliced products, constructs containing duplicated or rearranged 3' splice sites were created. In construct VP1,1, 160 bp surrounding the VP3 3' splice site was substituted with the corresponding region of the VP1 3' splice site. This construct resulted in the duplication of the VP1 3' splicing signal. VP3,3 (two identical VP3 3' splice sites) and VP1,3 (VP1 and VP3 3' splice sites reversed) were similarly created. Each construct maintained wild-type spacing between the 3' splice sites. Analysis of RNAs from transfections showed that in each construct, the 3' splice closest to the polyadenylation site was used preferentially. Analysis of a number of additional constructs indicated that there are no strong cis-acting positive or negative regulators of polyomavirus late splicing; rather, splicing choices appear to be determined largely by relative position of splice sites.
Insights
Polyomavirus late pre-mRNAs exhibit alternative splicing, producing distinct mRNA variants. Splicing choices are primarily dictated by the relative position of 3' splice sites, not their inherent strength.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Polyomavirus late pre-mRNAs undergo alternative splicing.
- This process generates multiple mRNA variants (mVP1, mVP2, mVP3) through differential usage of 3' splice sites.
- The relative abundance of these variants suggests a regulatory mechanism influencing splice site selection.
Purpose of the Study:
- To investigate whether splice site strength dictates the frequency of alternative splicing events in polyomavirus late pre-mRNAs.
- To identify potential cis-acting regulatory elements governing polyomavirus splicing choices.
Main Methods:
- Construction of engineered polyomavirus late gene expression vectors with duplicated or rearranged 3' splice sites.
- Analysis of RNA transcripts from transfected cells using RNA analysis techniques.
- Comparison of splicing patterns in constructs with altered splice site configurations.
Main Results:
- In all tested constructs, the 3' splice site located nearest to the polyadenylation signal was preferentially utilized.
- No strong positive or negative cis-acting regulatory elements were identified that significantly influenced polyomavirus late splicing.
- The observed alternative splicing patterns were largely determined by the positional arrangement of the 3' splice sites.
Conclusions:
- Splice site strength is not the primary determinant of alternative splicing ratios in polyomavirus late pre-mRNAs.
- The relative position of 3' splice sites to the polyadenylation site is the major factor controlling splicing decisions.
- Polyomavirus late splicing regulation relies on positional information rather than strong specific regulatory sequences.