Abstract
The primary mRNA sequence determines its secondary structure and the repertoire of interacting RNA-binding proteins (RBPs). The resulting mRNA ribonucleoprotein complex (mRNP) then influences all stages of the life of an mRNA. Here, we determined the mRNP composition of individual Kaposi sarcoma herpesviral (KSHV) mRNAs. Like all herpesviruses, KSHV switches between a latent and lytic stage of the viral life cycle. During reactivation from latency, the viral RNA regulator ORF57 ensures the translation of viral mRNAs by increasing their mRNA stability and nuclear export. We optimized an LNA/DNA mixmer RNA capture protocol for both transfection and viral infection settings. In combination with eCLIP, we confirmed that ORF57 directly binds to an AU-rich RNA motif, which may enable ORF57 to discriminate viral from cellular RNAs based on the nucleotide bias of KSHV lytic RNAs. In addition, we captured the RBPome of two ORF57-dependent viral transcripts and identified the host RNA processing factor SRSF3 as a key regulator of viral replication.
Keywords:
KSHV; ORF57; RNA-interactome capture; SRSF3; herpesviral gene expression.