Biotin-pgRNAWT or Biotin-pgRNAAUUUA to AUCUC RNA pulldown LC-MS/MS

ABSTRACT: The nuclear export of HBV RNAs allows the virus to synthesize its proteins through the translation machinery and replicate its genome through reverse transcription in the cytoplasm. However, the molecular mechanisms underlying this important process remain largely obscure. To illustrate this process, we took advantage of an unbiased HBV RNA-host protein interaction screen using a quantitative proteomics approach and identified embryonic lethal, abnormal vision, Drosophila-like 1 (ELAVL1) as a viral RNA binding partner. RNA scope and subcellular mRNA assays indicated that genetic and pharmaceutic inhibition of ELAVL1 inhibits HBV RNA nuclear export and suppresses viral replication in cell cultures. The observations of an HBV replication mouse model with ELAVL1 displayed similar results. RNA pulldown and RNA electrophoretic mobility shift assays revealed direct interaction between ELAVL1 and HBV pgRNA and confirmed AU-rich elements as the binding sites through site-directed mutagenesis of pgRNA. RNA-immunoprecipitation revealed that HBV RNAs associate with ELAVL1, which in turn binds to acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) and ANP32B. These interactions subsequently recruit CRM1. A nuclear RNase-targeted siRNA screen uncovered RNA exosome-mediated degradation of retardant HBV RNA after ELAVL1 or CRM1 knockdown. Further investigation revealed that ELAVL1 protects pgRNA from degradation. Notably, HBc deletion had no effect on pgRNA-CRM1 interaction and HBV RNA nuclear export. In summary, our work indicates that ELAVL1 functions both in HBV RNA stability and nucleocytoplasmic transport via the CRM1 nuclear export pathway.

INSTRUMENT(S): Orbitrap Exploris 480

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Yingcheng Zheng  

LAB HEAD: Yuchen Xia

PROVIDER: PXD044015 | Pride | 2024-05-21

REPOSITORIES: Pride