DHX34 interacts with mRNA processing complexes. (A) Cartoon depicting the CRISPR-mediated tagging of the endogenous DHX34 locus in HEK293T cells to generate carboxy-terminal tagged DHX34–GFP–FLAG cell lines. (B) Volcano plot of 125 common interacting proteins identified by mass spectrometry (Log2 ratio >2) for DHX34–GFP–FLAG A5, 1A10, and 1B3 CRISPR clones. Protein names are indicated for the top 50 enriched ribosomal (blue), spliceosomal (red) and EJC proteins (green). Due to space constraints, not all protein names are indicated in the plot. All identified proteins are listed in Supplemental Table 1. (C) String network of interacting proteins identified by mass spectrometry of anti-GFP immunopurifications from three independent CRISPR DHX34–GFP–FLAG clones. DHX34 interacts with protein complexes involved in mRNA biogenesis: spliceosome (red), EJC (green), and ribosome (blue). (D) Validation of mass spectrometry experiments with anti-GFP immunoprecipitations (IPs) of three different CRISPR clones used for mass spectrometry. Inputs and anti-GFP IPs were separated by SDS-PAGE and probed with the indicated antibodies in western blot assays. (E) U5 snRNA copurifies with DHX34, whereas two other snRNAs present in the spliceosomal complex C, U2, and U6, are not enriched in the IP. RNA–protein complexes were immunopurified using anti-FLAG beads from GFP–FLAG A5 clone, following an elution step; RNA was reverse-transcribed and PCR amplified with specific primers for spliceosomal snRNAs.
