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Polyoma virus small tumor antigen pre-mRNA splicing requires cooperation between two 3' splice sites
1Department of Biological Sciences, Columbia University, New York, NY 10027.
Abstract:
We have studied splicing of the polyoma virus early region pre-mRNA in vitro. This RNA is alternatively spliced in vivo to produce mRNA encoding the large, middle-sized (MTAg), and small (StAg) tumor antigens. Our primary interest was to learn how the 48-nucleotide StAg intron is excised, because the length of this intron is significantly less than the apparent minimum established for mammalian introns. Although the products of all three splices are detected in vitro, characterization of the pathway and sequence requirements of StAg splicing suggests that splicing factors interact with the precursor RNA in an unexpected way to catalyze removal of this intron. Specifically, StAg splicing uses either of two lariat branch points, one of which is located only 4 nucleotides from the 3' splice site. Furthermore, the StAg splice absolutely requires that the alternative MTAg 3' splice site, located 14 nucleotides downstream of the StAg 3' splice site, be intact. Insertion mutations that increase or decrease the quality of the MTAg pyrimidine stretch enhance or repress StAg as well as MTAg splicing, and a single-base change in the MTAg AG splice acceptor totally blocks both splices. These results demonstrate the ability of two 3' splice sites to cooperate with each other to bring about removal of a single intron.
Insights
Polyoma virus early region pre-mRNA splicing was studied in vitro. Findings reveal an unusual mechanism for excising a short intron, highlighting cooperation between two 3' splice sites.
Area of Science:
- Molecular Biology
- RNA Splicing Mechanisms
- Virology
Background:
- Polyoma virus early region pre-mRNA undergoes alternative splicing in vivo.
- This process generates mRNAs encoding large, middle-sized (MTAg), and small (StAg) tumor antigens.
- The StAg intron is unusually short, posing questions about its excision mechanism.
Purpose of the Study:
- To investigate the in vitro splicing of polyoma virus early region pre-mRNA.
- To elucidate the pathway and sequence requirements for the excision of the short StAg intron.
- To understand how splicing factors interact with precursor RNA for efficient intron removal.
Main Methods:
- In vitro splicing assays using polyoma virus early region pre-mRNA.
- Characterization of splicing products and intermediates.
- Analysis of sequence requirements through insertion and point mutations.
Main Results:
- All three predicted splice products were detected in vitro.
- StAg splicing utilizes alternative lariat branch points, one very close to the 3' splice site.
- StAg splicing absolutely requires the integrity of the downstream MTAg 3' splice site.
- Mutations affecting the MTAg pyrimidine stretch or splice acceptor impact both StAg and MTAg splicing.
Conclusions:
- Splicing factors interact with precursor RNA in a novel manner to facilitate StAg intron removal.
- The excision of the short StAg intron depends on the cooperation of two distinct 3' splice sites.
- This study demonstrates a mechanism where two 3' splice sites collaborate for efficient intron removal.