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cis-acting intron mutations that affect the efficiency of avian retroviral RNA splicing: implication for mechanisms
R A Katz1, M Kotler, A M Skalka
1Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111.
Abstract:
The full-length retroviral RNA transcript serves as (i) mRNA for the gag and pol gene products, (ii) genomic RNA that is assembled into progeny virions, and (iii) a pre-mRNA for spliced subgenomic mRNAs. Therefore, a balance of spliced and unspliced RNA is required to generate the appropriate levels of protein and RNA products for virion production. We have introduced an insertion mutation near the avian sarcoma virus env splice acceptor site that results in a significant increase in splicing to form functional env mRNA. The mutant virus is replication defective, but phenotypic revertant viruses that have acquired second-site mutations near the splice acceptor site can be isolated readily. Detailed analysis of one of these viruses revealed that a single nucleotide change at -20 from the splice acceptor site, within the original mutagenic insert, was sufficient to restore viral growth and significantly decrease splicing efficiency compared with the original mutant and wild-type viruses. Thus, minor sequence alterations near the env splice acceptor site can produce major changes in the balance of spliced and unspliced RNAs. Our results suggest a mechanism of control in which splicing is modulated by cis-acting sequences at the env splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing control.
Insights
Altering retroviral splice acceptor sites significantly impacts RNA splicing. Minor sequence changes near the env splice acceptor site can restore viral growth by modulating splicing efficiency.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Retroviral RNA transcripts serve multiple roles: mRNA for gag/pol, genomic RNA for virions, and pre-mRNA for spliced mRNAs.
- A precise balance of spliced and unspliced RNA is crucial for efficient retroviral virion production.
Purpose of the Study:
- To investigate the role of cis-acting sequences at the avian sarcoma virus env splice acceptor site in regulating RNA splicing.
- To explore how mutations affecting splicing impact retroviral replication and virion production.
Main Methods:
- Introduction of an insertion mutation near the env splice acceptor site of avian sarcoma virus.
- Isolation and characterization of phenotypic revertant viruses with second-site mutations.
- Detailed genetic and molecular analysis of revertant viruses to identify specific nucleotide changes and their effects on splicing.
Main Results:
- An insertion mutation near the env splice acceptor site led to increased splicing and defective viral replication.
- Second-site mutations, particularly a single nucleotide change at -20, restored viral growth.
- This single nucleotide change significantly decreased splicing efficiency compared to both mutant and wild-type viruses.
Conclusions:
- Minor sequence alterations at the env splice acceptor site can drastically alter the balance of spliced and unspliced RNAs.
- Splicing control in retroviruses can be modulated by cis-acting sequences at splice acceptor sites.
- This retroviral system offers a valuable genetic tool for studying mutations affecting RNA splicing control.