Fig. 7: USP39 facilitates efficient splicing of the oncogenic transcription factor HMGA2.

A Expression of individual HMGA2 transcript isoforms was analyzed using RNA-seq data for A2780 cells with USP39 knockdown (sh-USP39) and control cells (n = 3 biologically independent samples). B Sashimi plot visualization of RNA-seq reads mapping to HMGA2 in A2780 cells in response to USP39 knockdown. C Splicing efficiency analysis of HMGA2 at the 5′ and 3′ splicing sites upon USP39 depletion based on RNA-seq data. The red point represents HMGA2. Efficiency 5′ = transread count/ 5′ intron end first base coverage; Efficiency 3′ = transread count/ 3′ intron end last base coverage. D Pre-mRNA/mRNA ratio in A2780 cells with USP39 overexpression or knockdown (sh-USP39) was measured by q-PCR. E Schematic diagram showing HMGA2 minigene constructs and alternative splicing types. F The expression of HMGA2 minigene transcripts in Hela cells transfected HMGA2 minigenes in combination with USP39 siRNAs was measured by semi-quantitative PCR. G IGV tracks displaying the coverage of HMGA2 by RIP-seq reads. H The interaction between USP39 and the HMGA2 transcript was validated by RIP-PCR of A2780 cells overexpressing FLAG-USP39. FLAG antibody was used for immunoprecipitation. U6snRNA served as a positive control and FOXM1 as a negative control (n = 3 biologically independent samples). I RNA-pull down assay showing the interaction between the HMGA2 transcript and USP39 protein. HMGA2 site1 is an intronic sequence and the site2 sequence spanned an exon-intron junction. P value was obtained by Student’s t-test. Results represent the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.