Figure 3.
Pre-rRNAs are degraded upon depletion of DHX37 and expression of catalytically inactive DHX37 leads to defects in A’ cleavage and the conversion of 21S to 18SE. (a) C-terminally His6-tagged DHX37 or DHX37 carrying a threonine to alanine substitution at amino acid 282 within the evolutionarily conserved ‘GKT’ motif (DHX37T282A) was recombinantly expressed in E. coli and purified. Purified proteins were separated by SDS-PAGE and visualized by Coomassie staining. (b) The amount of ATP hydrolyzed by recombinant DHX37 or DHX37T282A in the presence (+) or absence (-) of RNA was determined using an in vitro NADH-coupled ATPase assay. Experiments were performed in triplicate and error bars represent mean ± standard deviation. (c) HEK293 cell lines were transfected with either non-target siRNAs (siNT) or siRNAs targeting DHX37 (siDHX37_1) and expression of the Flag tag, or C-terminally Flag-tagged DHX37 or DHX37T282A was induced by addition of tetracycline 24 h before harvesting. Proteins were extracted and analyzed by western blotting using antibodies against DHX37, tubulin and the Flag tag. (d) Total RNA extracted form HEK293 cell lines treated as described in (c) was separated by denaturing agarose gel electrophoresis, transferred to a nylon membrane and analyzed by northern blotting using probes hybridizing at the 5ʹ end of ITS1 (left panel) or within the 5ʹ ETS (right panel). Pre-rRNAs were detected using a phosphorimager and the mature 28S rRNA was visualized by methylene blue staining (MB).