Figure 5.
The U3 snoRNA accumulates on pre-ribosomes upon expression of catalytically inactive DHX37. (a) HEK293 cells capable of expression of DHX37-Flag or DHX37T282A-Flag were transfected with siRNAs against DHX37 and 24 h prior to harvesting, expression of the tagged proteins was induced by addition of tetracycline. To confirm equal expression levels, proteins were analyzed by western blotting using antibodies against DHX37 and tubulin. (b) Whole cell extracts prepared from HEK293 cells treated as in (a) were separated by sucrose density gradient centrifugation. The optical density of each faction at 260 nm was determined and used to generate a profile on which the peaks corresponding to ribosomal and pre-ribosomal complexes are indicated. RNA extracted from the gradient fractions described in (b) was separated by denaturing PAGE and transferred to a nylon membrane. Northern blotting was performed using a [32P]-labelled probe hybridizing to the U3 snoRNA. The experiments shown in this figure were performed in triplicate and representative data are shown.