Human regulatory proteins associate with non-coding RNAs from the EBV IR1 region

Abstract

Objective: The function of Epstein-Barr virus (EBV) stable intronic sequence (sis)RNAs, non-coding RNAs transcribed from a region required for EBV-mediated cellular transformation, remain unknown. To better understand the function of ebv-sisRNA-1 and ebv-sisRNA-2 from the internal repeat (IR)1 region of EBV, we used a combination of bioinformatics and biochemistry to identify associated RNA binding proteins. The findings reported here are part of ongoing studies to determine the functions of non-coding RNAs from the IR1 region of EBV.

Results: Human regulatory proteins HNRNPA1 (heterogeneous nuclear ribonucleoprotein A1), HNRNPC, HNRNPL, HuR (human antigen R), and protein LIN28A (lin-28 homolog A) were predicted to bind ebv-sisRNA-1 and/or ebv-sisRNA-2; FUS (fused in sarcoma) was predicted to associate with ebv-sisRNA-2. Protein interactions were validated using a combination of RNA immunoprecipitation and biotin pulldown assays. Both sisRNAs also precipitated with HNRNPD and NONO (non-POU domain-containing octamer-binding protein). Interestingly, each of these interacting proteins also precipitated non-spliced non-coding RNA sequences transcribed from the IR1 region. Our findings suggest interesting roles for sisRNAs (through their interactions with regulatory proteins) and provide further evidence for the existence of non-spliced stable non-coding RNAs.

Keywords: EBNA-LP; EBV; FUS; HNRNP; HuR; IR1; LIN28; NONO; lncRNA; ncRNA; sisRNA.

Fig. 1

Interaction of ebv-sisRNA-1 with HNRNPL and HNRNPD. a Schematic illustration of the structure of ebv-sisRNA-1. Base pairing is depicted by lines and circles. The mutations present in the ΔCA form are indicated. b Silver stained gel after biotinylated precipitation from whole BJAB lysate. The ebv-sisRNA-1 wild-type (sisRNA-1wt) form was compared to either a randomized sequence, which represents background non-specific RNA binding proteins, or the mutated ΔCA form. Bands excised and subjected to mass spectrometry are denoted with asterisks. c Fold enrichment of ebv-sisRNA-1 following RIP using antibodies against HNRNPL, HNRNPD, or normal rabbit IgG from either BJAB-B1 or Raji cells. Data represent the mean of two independent RIPs per cell line (one for BJAB-B1 HNRNPD) and are normalized to control IgG. See Additional file 3 for individual RIP data point values

Fig. 2

RNA protein binding profile from one W-repeat of the IR1 region. a Schematic illustration of the EBNA-LP locus (top) with internal W-repeats. The region used for RBPmap [25] is enhanced (bottom, at scale) and shows the W1 and W2 exons compared to the sisRNA (s1 or s2) introns. Regions amplified for PCR (amplicon) in these studies are shown in purple: a1—ebv-sisRNA-1, a2—ebv-sisRNA-2, a3—sisRNA-1 to W2, a4—W1 to W2, and a5—across both exons. b RNA binding proteins resulting from RBPmap are indicated by region (above, not to scale). Blue indicates proteins assayed by RIP in addition to HNRNPL (bold). c Top results from STRING [38] analysis for enrichment in biological process (top) and KEGG pathway; false discovery rate (FDR) is given. d Fold enrichment of ebv-sisRNA-2 after RIP using HNRNPL, HNRNPD, or normal rabbit IgG antibodies and either BJAB-B1 or Raji cells. Data represent the mean of two independent RIPs per cell line (one for BJAB-B1 HNRNPD) and are normalized to control IgG. See Additional file 3 for individual RIP data point values

Fig. 3

RIP binding of ncRNA from EBV W-repeat region. a Fold enrichment for ebv-sisRNA-1 or ebv-sisRNA-2 after RIP using antibodies against the indicated proteins from BJAB-B1 or Raji cells. b Fold enrichment for ebv-sisRNA-1 to exon W2 (a3 from Fig. 2a) after RIP using antibodies against the indicated proteins from BJAB-B1 or Raji cells. For all RIPs, data represent the mean of mostly two independent experiments per cell line (where only one experiment was done is shown in Additional file 3) and data are normalized to control IgG. For both A and B, data are cut off at a fold enrichment value of 25. c RT-PCR (a4 from Fig. 2a) from either lentiviral transduced BJAB using empty vector or a W1 through W2 overexpression (OE) construct or from two independent RNA isolations from BJAB-B1 cells. The amplified forms of the unspliced (u) or spliced (s) products are indicated. d RT-PCR (a5 from Fig. 2a) from BJAB, BJAB-B1, or Raji total RNA across the exon/intron boundaries (a5 from Fig. 2a) for retention of sisRNA-1. The amplified forms of the unspliced (u) or spliced (s) products are indicated