A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro
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Discussion
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Implication
In vitro study reveals that protein factor ASF increases small t mRNA splicing in kidney cells, indicating cell-specific regulation of alternative splice-site selection.
Key Points
- To identify and characterize the molecular factor driving the cell-specific alternative splicing of SV40 early pre-mRNA observed in human embryonic kidney 293 cells.
- Compared alternative splicing patterns of SV40 early pre-mRNA in vitro using nuclear extracts prepared from human embryonic kidney 293 cells and HeLa cells.
- Developed an in vitro complementation assay to monitor and purify the splicing regulatory activity from 293 cell nuclear extracts.
- Characterized the factor's molecular composition using heat treatment and micrococcal nuclease digestion assays.
- Nuclear extracts from 293 cells demonstrated a pronounced preference for small t mRNA splicing compared to extracts derived from HeLa cells.
- Purification isolated an alternative splicing factor (ASF) of 30 to 35 kDa that markedly increased small t splicing with a concomitant reduction in large T splicing.
- The isolated factor was sensitive to heat inactivation but resistant to micrococcal nuclease, showing it is a protein factor that lacks an accessible, essential RNA component.
Cite This Study
Ge et al. (1990) studied this question.