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Inhibition of RNA splicing at the Rous sarcoma virus src 3' splice site is mediated by an interaction between a
B A Amendt1, S B Simpson, C M Stoltzfus
1Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Abstract:
In permissive Rous sarcoma virus-infected chicken embryo fibroblasts (CEF), approximately equimolar amounts of env and src mRNAs are present. In nonpermissive mammalian cells, the src mRNA level is elevated and env mRNA level is reduced. A cis element in the region between the env gene and the src 3' splice site, which we have termed the suppressor of src splicing (SSS), acts specifically in CEF but not in human cells to reduce src mRNA levels. The splicing inhibition in CEF is not caused by a base-paired structure which is predicted to form between the SSS and the src 3' splice site. To further investigate the mechanism of the inhibition, we have used human HeLa cell nuclear extracts to compare in vitro the rates of splicing of RNA substrates containing the Rous sarcoma virus major 5' splice site and either the env or src 3' splice sites. We show that the src 3' splice site is used approximately fivefold more efficiently than the env 3' splice site. The efficiency of in vitro splicing at the src 3' splice site is specifically reduced by addition of CEF nuclear extract. The inhibition is dependent on the presence of the SSS element and can be abrogated by addition of competitor RNA. We propose that the SSS region represents a binding site for a negative-acting CEF splicing factor(s).
Insights
A suppressor of src splicing (SSS) element in chicken cells inhibits src mRNA levels by binding a negative splicing factor. This mechanism, absent in human cells, explains differential gene expression in Rous sarcoma virus infections.
Area of Science:
- Molecular Biology
- Virology
- Gene Regulation
Background:
- Rous sarcoma virus (RSV) infection leads to differential mRNA levels of env and src genes in permissive chicken embryo fibroblasts (CEF) versus nonpermissive mammalian cells.
- Elevated src mRNA and reduced env mRNA in mammalian cells suggest regulatory mechanisms controlling viral gene expression.
Purpose of the Study:
- To investigate the mechanism by which a cis element, the suppressor of src splicing (SSS), specifically reduces src mRNA levels in CEF but not in human cells.
- To elucidate the role of the SSS element in regulating alternative 3' splice site usage in RSV-infected cells.
Main Methods:
- In vitro splicing assays using RNA substrates with Rous sarcoma virus 5' splice site and either env or src 3' splice sites.
- Comparison of splicing efficiency in human HeLa cell nuclear extracts with and without the addition of CEF nuclear extracts.
- Analysis of the effect of the SSS element and competitor RNA on splicing inhibition.
Main Results:
- The src 3' splice site is utilized approximately fivefold more efficiently than the env 3' splice site in vitro.
- Chicken embryo fibroblast nuclear extract specifically inhibits in vitro splicing at the src 3' splice site, dependent on the SSS element.
- Inhibition of src splicing by CEF extract is abrogated by competitor RNA, suggesting a protein-binding mechanism.
Conclusions:
- The SSS region acts as a binding site for a negative-acting chicken embryo fibroblast splicing factor(s).
- This factor-mediated regulation of splicing efficiency at the src 3' splice site contributes to differential gene expression in Rous sarcoma virus-infected chicken cells.
- The absence of this regulatory mechanism in human cells explains the distinct viral gene expression patterns observed in permissive versus nonpermissive hosts.