Analysis of a structured intronic region of the LMP2 pre-mRNA from EBV reveals associations with human regulatory proteins and nuclear actin

Abstract

Objective: The pre-mRNA of the Epstein-Barr virus LMP2 (latent membrane protein 2) has a region of unusual RNA structure that partially spans two consecutive exons and the entire intervening intron; suggesting RNA folding might affect splicing - particularly via interactions with human regulatory proteins. To better understand the roles of protein associations with this structured intronic region, we undertook a combined bioinformatics (motif searching) and experimental analysis (biotin pulldowns and RNA immunoprecipitations) of protein binding. Result: Characterization of the ribonucleoprotein composition of this region revealed several human proteins as interactors: regulatory proteins hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1), hnRNP U, HuR (human antigen R), and PSF (polypyrimidine tract-binding protein-associated splicing factor), as well as, unexpectedly, the cytoskeletal protein actin. Treatment of EBV-positive cells with drugs that alter actin polymerization specifically showed marked effects on splicing in this region. This suggests a potentially novel role for nuclear actin in regulation of viral RNA splicing.