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Evidence for a HeLa cell splicing activity that is necessary for activation of a regulated adenovirus 3' splice site
K Zerivitz1, J P Kreivi, G Akusjärvi
1Department of Microbial Genetics, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
Abstract:
Adenovirus late region 1 pre-mRNA splicing is temporally regulated during a lytic infection at the level of alternative 3' splice site usage to produce two mRNAs; the 52,55K and IIIa mRNAs which utilize the proximal and distal 3' splice sites, respectively. In vivo, the 52,55K mRNA is produced both early and late after infection, while IIIa is produced exclusively late in infection. Uninfected HeLa cell nuclear extracts, prepared with a low salt (0.4-0.5 M) or high salt (0.6 M and higher) wash, differed in their ability to splice 52,55K, IIIa and beta-globin transcripts. 52,55K and beta-globin precursors were spliced with similar efficiency in the low and high salt extract, while the IIIa mRNA was generated only in the high salt extract. Using the beta-globin pre-mRNA, no kinetic differences between the two types of extracts were observed. Nor were there any significant differences in the snRNA composition. The IIIa splicing activity did not appear to correlate with U2AF and pPTB levels. Our results suggest that a cellular trans-acting factor(s), which is required for adenovirus IIIa 3' splice site activation, is solubilized only at high salt concentrations.
Insights
Adenovirus splicing of IIIa mRNA requires a specific cellular factor only found in high-salt nuclear extracts. This factor, essential for late-stage viral gene expression, is not present in low-salt extracts, revealing temporal regulation mechanisms.
Area of Science:
- Molecular Biology
- Virology
- Gene Expression Regulation
Background:
- Adenovirus late region 1 pre-mRNA splicing is temporally regulated during lytic infection.
- Alternative 3' splice site usage produces 52,55K (proximal site) and IIIa (distal site) mRNAs.
- 52,55K mRNA is produced early and late, while IIIa mRNA is exclusively late.
Purpose of the Study:
- To investigate the differential splicing regulation of adenovirus late region 1 pre-mRNAs.
- To identify cellular factors involved in the temporal control of IIIa mRNA production.
- To understand the role of nuclear extract salt concentrations in spliceosome activity.
Main Methods:
- Preparation of HeLa cell nuclear extracts with varying salt concentrations (low: 0.4-0.5 M; high: ≥0.6 M).
- In vitro splicing assays using adenovirus 52,55K and IIIa pre-mRNAs, and beta-globin pre-mRNA.
- Analysis of splicing efficiency, kinetics, snRNA composition, and protein levels (U2AF, pPTB).
Main Results:
- High-salt nuclear extracts specifically facilitated the splicing of adenovirus IIIa pre-mRNA, unlike low-salt extracts.
- Splicing of 52,55K and beta-globin pre-mRNAs occurred efficiently in both low and high-salt extracts.
- IIIa splicing activity did not correlate with U2AF or pPTB levels, suggesting a distinct regulatory factor.
Conclusions:
- A cellular trans-acting factor(s) is required for adenovirus IIIa 3' splice site activation.
- This essential factor is only solubilized and active at high salt concentrations.
- High-salt-dependent factor solubilization explains the late-stage-specific production of IIIa mRNA.