Intronic sequences and 3' splice sites control Rous sarcoma virus RNA splicing

Area of Science:

• Molecular Biology
• Virology
• RNA Splicing Mechanisms

Background:

• Incomplete splicing is a critical aspect of the retroviral life cycle.
• Rous sarcoma virus (RSV) RNA splicing involves cis-acting regulatory elements that control splicing efficiency.

Purpose of the Study:

• To investigate the cis-acting sequences of RSV RNA that regulate incomplete splicing.
• To elucidate the roles of 5' and 3' splice sites and intronic negative regulatory elements in splicing inhibition.

Main Methods:

• Chimeric constructs containing RSV splice sites and regulatory elements were created.
• Transient transfection experiments were used to evaluate splicing inhibition.
• Sequence deletions and insertions were performed to assess functional roles.

Main Results:

• RSV 5' splice site was efficiently used, but RSV env and src 3' splice sites showed incomplete splicing (40-50% unspliced RNA).
• An intronic negative regulator of splicing element enhanced inhibition (70-80% unspliced RNA) and acted additively with 3' splice sites.
• Deletion of upstream sequences near the src 3' splice site restored efficient splicing, indicating upstream elements cause inefficient usage.

Conclusions:

• RSV splicing control is mediated by both intronic sequences and 3' splice sites.
• Distinct mechanisms contribute to the underutilization of env and src splice acceptor sites.
• Upstream sequences and potential secondary structure interactions play a significant role in regulating RSV splicing efficiency.