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Regulation of adenovirus alternative RNA splicing at the level of commitment complex formation
1Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
Abstract:
The adenovirus late region 1 (L1) represents an example of an alternatively spliced gene where one 5' splice site is spliced to two alternative 3' splice sites, to produce two mRNAs; the 52,55K and IIIa mRNAs, respectively. Accumulation of the L1 mRNAs is temporally regulated during the infectious cycle. Thus, the proximal 3' splice site (52,55K mRNA) is used at all times during the infectious cycle whereas the distal 3' splice site (IIIa mRNA) is used exclusively late in infection. Here we show that in vitro splicing extracts prepared from late adenovirus-infected cells reproduces the virus-induced temporal shift from proximal to distal 3' splice site selection in L1 pre-mRNA splicing. Two stable intermediates in spliceosome assembly have been identified; the commitment complex and the pre-spliceosome (or A complex). We show that the transition in splice site activity in L1 alternative splicing results from an increase in the efficiency of commitment complex formation using the distal 3' splice site in extracts prepared from late virus-infected cells combined with a reduction of the efficiency of proximal 3' splice site splicing. The increase in commitment activity on the distal 3' splice site is paralleled by a virus-induced increase in A complex formation on the distal 3' splice site. Importantly, the virus-induced shift from proximal to distal L1 3' splice site usage does not require cis competition between the 52,55K and the IIIa 3' splice sites, but rather results from the intrinsic property of the two 3' splice sites which make them respond differently to factors in extracts prepared from virus-infected cells.
Insights
Adenovirus alternative splicing temporally regulates gene expression. During infection, the virus shifts 3' splice site selection in L1 pre-mRNA, favoring distal sites late in infection.
Area of Science:
- Molecular Biology
- Virology
- Gene Regulation
Background:
- Adenovirus late region 1 (L1) exhibits alternative splicing, producing 52,55K and IIIa mRNAs from a single 5' splice site and two alternative 3' splice sites.
- L1 mRNA accumulation is temporally regulated during adenovirus infection, with proximal 3' splice site usage occurring throughout infection and distal 3' splice site usage exclusively late in infection.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the virus-induced temporal shift in 3' splice site selection in adenovirus L1 pre-mRNA splicing.
- To determine if in vitro splicing extracts from infected cells can replicate the observed temporal regulation of splice site usage.
Main Methods:
- Utilized in vitro splicing extracts prepared from adenovirus-infected cells.
- Analyzed spliceosome assembly intermediates, specifically the commitment complex and pre-spliceosome (A complex).
- Assessed the efficiency of splice site selection for both proximal and distal 3' splice sites.
Main Results:
- In vitro splicing extracts from late adenovirus-infected cells reproduced the temporal shift in L1 3' splice site selection.
- The shift is attributed to increased efficiency of commitment complex formation at the distal 3' splice site and reduced efficiency at the proximal site in late-infected cell extracts.
- Virus-induced increases in A complex formation at the distal 3' splice site correlate with the shift in usage.
- The observed shift does not depend on cis competition between the two 3' splice sites.
Conclusions:
- Adenovirus infection alters the cellular splicing machinery to promote the use of the distal 3' splice site (IIIa mRNA) late in infection.
- The temporal regulation of L1 alternative splicing is mediated by changes in the efficiency of spliceosome assembly intermediates, particularly commitment complex formation.
- The intrinsic properties of the alternative 3' splice sites, rather than cis competition, dictate their differential usage in response to viral factors.