An RNA splicing enhancer-like sequence is a component of a splicing inhibitor element from Rous sarcoma virus

Area of Science:

• Molecular Biology
• Virology
• RNA Splicing

Background:

• Retrovirus replication involves accumulating unspliced viral RNA for mRNA and genomic functions.
• The negative regulator of splicing (NRS) in Rous sarcoma virus inhibits splicing to facilitate this accumulation.
• The NRS contains two critical sequences: one for U11 small nuclear ribonucleoprotein (snRNP) binding and a purine-rich region interacting with splicing factor SF2/ASF.

Purpose of the Study:

• To investigate if the purine-rich region of the NRS functions as an RNA splicing enhancer.
• To determine the role of SF2/ASF interaction in the NRS-mediated splicing inhibition.

Main Methods:

• In vitro splicing assays using Drosophila doublesex pre-mRNA.
• Site-directed mutagenesis and deletion analysis of the NRS.
• Chimera construction by substituting authentic enhancers (ASLV, FP) for the NRS purine region.
• Analysis of splicing inhibition in transfected cells.

Main Results:

• The NRS demonstrated potent, orientation-dependent splicing enhancer activity in vitro, localized to the purine-rich domain.
• Enhancer activity correlated with SF2/ASF binding and efficient splicing inhibition.
• Substituting authentic enhancers restored splicing inhibition, indicating the functional significance of enhancer activity.
• A non-functional FP enhancer mutant (FPD) that doesn't bind SF2/ASF failed to inhibit splicing, supporting the role of SF2/ASF.

Conclusions:

• The purine-rich region of the NRS acts as an RNA splicing enhancer.
• SF2/ASF binding to the NRS is crucial for splicing inhibition, although other SR proteins may compensate.
• The NRS likely facilitates snRNP binding via SF2/ASF, contributing to unspliced viral RNA accumulation.