Fig 1: PDAP1 protein and RNA interactions.(A) (Top) SDS-PAGE gel of anti-Flag precipitates from lysates of HAV-infected cells transfected with the empty vector versus PDAP1-Flag expression vector. (Bottom) Volcano plot of proteins identified by LC-MS in in-gel digests of anti-Flag precipitates. (B) STRING v12.0 prediction of physical complexes formed by proteins >8-fold enriched in anti-Flag precipitates from cells with or without HAV infection [shaded zone in (A)]. The thickness of connecting lines corresponds to confidence of the physical interaction. Proteins associated with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway for ribosome biogenesis are shown in green (hsa03008, Q = 2.53 × 10−5), spliceosome in blue (hsa03040, Q = 1.69 × 10−6) or Gene Ontology (GO) for large ribosome subunit biogenesis in magenta (GO 0042273, Q = 5.20 × 10−9), and stress granule regulation in yellow (GO 0063029, Q = 7.8 × 10−4) or green (GO 1903608, Q = 5.55 × 10−5). (C) Immunoblots of anti-Flag precipitates from lysates of cells transfected with the PDAP1-Flag expression vector or empty vector. A postribosome 100K supernatant and ribosome-enriched pellet were generated by high-speed centrifugation. L13A, large ribosomal subunit protein uL13. (D) Coimmunoprecipitation of recombinant C-terminally His-tagged rPDAP1-His and N-terminally His-tagged rHis-eIF1A, both produced in bacteria, with anti-PDAP1 or anti-eIF1A. Input mixtures contained a >300-fold molar excess of human serum albumin. (E) HAV circRNA IRES reporter activity in cells transfected with four siRNAs targeting eIF1A transcripts. Bars show GFP normalized to circRNA abundance. N = 3 experiments. P values by ANOVA. Below are immunoblots for GFP, eIF1A, and GAPDH (loading control) from a representative experiment. AU, arbitrary units. (F) NLuc activity 5 days after quasi-enveloped 18f-NLuc reporter virus infection of cells transfected previously with siRNAs targeting eIF1A. Immunoblots for eIF1A and β-actin (loading control) are shown below. LU, light units. (G) Immunoblots of proteins in lysates of sgCtrl and PDAP1-KO1.4 cells coprecipitating with an RNA probe comprising the complete 5′UTR of 18f virus linked to biotin at its 3′ end.