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.

Keywords: Actin; EBV; Epstein–Barr virus; Herpes virus; HuR; LMP2; PSF; RNA; Splicing; hnRNP A1; hnRNP U.

Fig. 1

Validation of protein interactors. a Secondary structure model of the intronic sequence. hnRNP A1, hnRNP U and HuR binding sites predicted by RBPmap are color coded. b Fold enrichment of the LMP2 pre-mRNA following RIPs carried out with antibodies against hnRNP A1, hnRNP U, hnRNP L, HuR, PSF and NONO. c Fold enrichment of pre-mRNA (junction spanning primers) and intron (internal primers) following RIPs with anti-actin antibody. Data represents the average (with standard deviation) of independent experiments all normalized to control RIP with IgG. All RIPs (except for RIPs for NONO and hnRNP L) were carried out as either 2 or 3 independent experiments. All primer sequences used for the experiments are included in Additional file 6: Table S4

Fig. 2

RT-PCR and qPCR analysis of spliced and unspliced transcripts following disruption of actin polymerization in BJAB B1 cells. a A cartoon of LMP2B with locations of exons RT-PCR primer sites and a model of the structured region is at the top. Below this are the results of RT-PCR analyses of spliced and unspliced transcripts in the presence of Latrunculin. b Spliced and unspliced variants quantified by qPCR analysis. Data shown are first normalized to housekeeping gene HPRT and plotted as a fold difference compared to the control at each time point. All data represents the mean (with standard deviation) from two independent experiments (*p < 0.05). All primer sequences used for the experiments are included in Additional file 6: Table S4