The CTD of DHX37 is required for interaction with UTP14A in vitro and for ribosome biogenesis in vivo. (A) Pull-down experiment using recombinant maltose-binding protein-tagged UTP14Amin and full-length and ΔCTD MmDHX37 expressed in HEK293 cells. (B) Immunofluorescence analysis of the localization of the 40S subunit assembly factor ENP1 in HeLa cells expressing wild-type EGFP-MmDHX37, a truncated version lacking the carboxy-terminal domain (ΔCTD), or the ATPase-deficient E369Q mutant. Expression of DHX37 proteins was induced with tetracycline for 24 h. Ninety minutes before fixation, cells were either supplemented with a solvent control or with 20 nM leptomycin B (LMB) to prevent CRM1-dependent nuclear export, leading to nucleoplasmic accumulation of ENP1 along with newly made 40S subunits in control cells (Badertscher et al. 2015). Note that the E369Q mutant prevents relocalization of ENP1 to the nucleoplasm in the presence of LMB, indicative of early 40S subunit maturation defects. (C) The CTD of DHX37 is required to rescue early 40S biogenesis defects upon DHX37 depletion. DHX37 was depleted from HeLa cells by CRISPRi. HeLa cells were left untreated (−) or transfected for 72 h with a plasmid encoding HA-tagged dCas9 and either a sgRNA targeting endogenous DHX37 (DHX37i) or a control sgRNA and transfected cells were selected with puromycin. Expression of the indicated EGFP-MmDHX37 rescue constructs was induced for 24 h with tetracycline. Cells were treated with LMB for 90 min before fixation as indicated. The expression of HA-tagged dCas9 and localization of the 40S assembly factor ENP1 were analyzed by immunofluorescence. Scale bars, 20 µM.
