Identification of host RNAs that interact with EBV noncoding RNA EBER2

Abstract

Epstein-Barr virus (EBV) expresses an abundant nuclear noncoding RNA called EBER2, which interacts with and acts as a guide RNA for the host transcription factor PAX5. This ribonucleoprotein complex localizes to the terminal repeat (TR) regions of the EBV genome via RNA-RNA interactions between EBER2 and nascent transcripts originating from these target sites. Given the fact that EBER2 base pairs with a viral RNA, we developed a protocol to identify EBER2-interacting RNAs in a transcriptome-wide manner. Our approach entails psoralen-mediated crosslinking, selection with antisense oligonucleotides targeting EBER2, and RNase V1 digestion coupled to next-generation sequencing. The use of RNase V1 circumvents the need of extensive computational analysis post data acquisition to search for predicted RNA hybrids, as the RNase V1 cleavage site marks the region of RNA duplex formation. As proof of principle, we show that our approach correctly identifies the known EBER2 interaction with TR RNAs. Moreover, we identify the host functional noncoding RNAs MRP, H1, and 7SL RNAs as well as three putative enhancer RNAs as candidate EBER2-interacting RNAs. As all of these gene loci exhibit PAX5 occupancy, we propose that EBER2 is recruited to these sites through its binding partner PAX5 and forms RNA-RNA interactions with nascent transcripts on chromatin. Thus, our novel approach facilitates the identification of targeted RNA-RNA-interactions and minimizes the need of downstream computational analyses to predict RNA duplexes.

Mapping of Influenza Virus RNA-RNA Interactions Reveals a Flexible Network

“…Reaction was cleaned up by washing beads once with PXL and twice with PNK buffer followed by an overnight RNA ligation reaction (10 mL 10x T4 RNA ligase 1 buffer, 10 mL 10 mM ATP, 5 mL T4 RNA ligase 1 (NEB), 2 mL RNasin, 73 mL H 2 O). Beads were washed twice with PXL and twice with PNK buffer, and Proteinase K-treated for 1 h at 50 C. Phenol-chloroform was added to the beads; the supernatant was collected and irradiated for 8 min with 254 nm UV light on ice to reverse AMT-crosslinks (Nanni and Lee, 2018). RNA was isolated by ethanol precipitation in the presence of 5 mg glycogen and resuspended in 10 mL H 2 O. Illumina-compatible deep sequencing library was constructed using the NEBNext Ultra II RNA Library Prep Kit (NEB).…”

5-methylcytosine modification of an Epstein–Barr virus noncoding RNA decreases its stability

“…This unbiased detection method requires ∼100 ng of highly pure input material devoid of common contaminating cellular RNAs, such as rRNAs or tRNAs, which contain a plethora of RNA modifications that would skew the analysis. We utilized a previously developed protocol to isolate EBERs from total RNA of EBV-positive lymphocytes (BJAB-B1 cells) by combining ASO selection under denaturing conditions and elution of bound RNAs with tetraethylammonium chloride (TEACl)-containing buffer (Nanni and Lee 2018), followed by PAGE purification (Fig. 1A,B).…”

Transcriptome analysis reveals significant differences between primary plasma cell leukemia and multiple myeloma even when sharing a similar genetic background

“…In our study, an unexpected finding was the deregulation of the EBV infection pathway as revealed by the KEGG analysis for gene, isoform and alternatively spliced exon expressions. In addition, a few studies have reported the use of cellular spliceosome machinery by EBV for the transcription of viral proteins in EBV-infected cells with splicing components 42,43 . We can speculate that the infection with EBV might lead to the overexpression of the WARS gene as an antiviral defence of the EBV-infected cells.…”