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.
Keywords: Epstein-Barr virus; RNA-RNA interaction; next-generation sequencing; noncoding RNA.