a, b, Depletion of the DDX ATPase UAP56 leads to an increase in nuclear speckle size. This is consistent with the model that UAP56, which does not contain LCDs and is not an essential ‘building block’ for nuclear speckles, is required for RNA turnover in speckles, and its absence would thus lead to an increased residence time of RNA in the compartment and a subsequent increase in the size of pre-existing compartments. a, A549 cells were transfected with control siRNA or UAP56 siRNA that targets the 3′ untranslated region. After 48 h, cells were infected with influenza virus (strain WSN) at a multiplicity of infection of 10 for 6 h. Cells were subjected to single-molecule RNA FISH to label viral M mRNA, and nuclear speckles were detected by immunostaining with an anti-SON antibody. ‘Viral M RNA’ is an influenza virus transcript that has been described to traffic through nuclear speckles22 and is used as a model to represent poly-adenylated, spliced cellular transcripts. Insets denote enlargements of the white squares. Scale bar, 10 μm. Images are representative of three independent experiments. b, The percentage of viral M mRNA at nuclear speckles was plotted against the nuclear speckle volume (423 nuclear speckles for each condition—control and UAP56 siRNA. c, Stably transfected A549 cells expressing wild-type or UAP56 mutant (E197A) were treated with UAP56 siRNA, and cells were subjected to immunostaining with an anti-SON antibody. Scale bars, 10 μm. Images are representative of three independent experiments. d, e, Selection and characterization of stably transfected A549 cells expressing wild-type or mutant (E197A) UAP56. For gel source data, see Supplementary Fig. 1. Images are representative of three independent experiments. d, Several cell clones stably expressing wild-type or mutant UAP56 were tested by western blot analysis using an anti-Flag antibody. Clone 2 of wild-type UAP56, and clone 3 of mutant UAP56 were selected for further studies. e, Immunofluorescence with an anti-Flag antibody shows similar expression levels of exogenous UAP56 or UAP56(E197A) in the selected stable cell lines. Scale bar, 10 μm. f, Merged images with nuclear envelope marker protein staining for experiments in Fig. 3f. Images are representative images of five (Sub2-degron) or six (Sub2) biological replicates. g, Cells were treated as in Fig. 4g. At time point t = 60 min after induction of reporter, induction cells were treated with either water or 5% 1.6 hexanediol for 20 min. Images are representative of three biological replicates. h, Quantification of the percentage of cells displaying either distinct nuclear RNA foci (transcription foci, TF; up to two to account for mitotic cells) or diffuse nuclear RNA signal in Sub2-depleted cells. Images are representative of five biological replicates with n > 380 cells per replicate. ***P = 0.0009, **** P < 0.0001, two-tailed unpaired t-test. Data are mean and s.e.m. Dots represent the mean of individual replicates.