RNA interactome capture identifies key proteins for herpesviral gene expression
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. In KSHV, the viral RNA regulator ORF57 ensures the translation of viral mRNAs by increasing mRNA stability and nuclear export. By optimizing an LNA/DNA mixmer RNA capture protocol to both transfection and virus replication settings, we identified the RBPome of specific ORF57-dependent viral transcripts. Both capture and eCLIP experiments robustly detected ORF57 as a direct RNA binder to an AU-rich motif, which may enable ORF57 to discriminate viral from cellular RNAs. Furthermore, we identified the RNA processing factor SRSF3 as a key regulator of viral replication. This work facilitates RNA-interactome studies of specific mRNAs and sheds light on how the mRNP composition orchestrates gene expression. Overall design: Enhanced crosslinking and immunoprecipitation (eCLIP) for RBM42 upon DNA damage and mock treatment on HCT116 cells. For each treatment we performed 2 replicates and comapred the eCLIP to size match input control