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RNA interactome capture identifies key proteins for herpesviral gene expression

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. 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE253314 | GEO | 2025/08/20

REPOSITORIES: GEO

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Project description:The Kaposi's sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi's sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman's disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exonintron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation. HITS-CLIP was performed on TREx BCBL-Rta cells 20 hpi using antibodies against ORF57. Three biological replicates were performed.

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RNA interactome capture identifies key proteins for herpesviral gene expression

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