Fig. 1

Global lengthening of 3′UTRs in DN identified from RNA-seq data and qRT-PCR verification. a The workflow for the identification of dynamic APA events from glomerular RNA-seq data. b PCA analysis of PDUI (top) and DPAU (bottom) score clearly separated DN patients from control subjects. c Scatter plots of median PDUI (top) or DPAU (bottom) scores between DN and control for each gene. Dashed lines represent ± 0.1 cutoffs for PDUI and ± 10 cutoffs for DPAU, respectively. The different 3′UTR isoforms resulted from the usage of distal PAS (red) or proximal PAS (blue) in DN were colored. d The volcano plots of 3ʹUTR lengthening (red) and shortening (blue) genes. The cut off value was ∆PDUI (DN-C) = ± 0.1 and -log10 (P-value) = 1.301 (1.301 corresponds to P-value = 0.05) for DaPars algorithm (top), ∆DPAU (DN-C) = ± 10 and -log10 (P-value) = 1.301 for QAPA algorithm (bottom). e The histogram showed the number of genes with 3ʹUTR lengthening and shortening due to APA. f Two representative RNA-seq tracks of dynamic APA-regulated genes (CYB5R1, PDLIM1) to highlight the 3′UTR coverage differences between DN and control samples. Purple track represented DN and blue track represented control. The red box indicated the different part of distal 3ʹUTR. g–h, The quantification of PDUI (g) and DPAU (h) changes for CYB5R1 (top) and PDLIM1 (bottom) between DN and control samples. i qRT-PCR analysis to verify the changes of distal and proximal PASs usage of CYB5R1 and PDLIM1 between DN and control. Schematic diagrams of the primer pair design were illustrated in left panel. The increased usage of distal PASs (L/T) in DN compared with control were quantified (middle and right panel). The data represented the mean ± SEM of six independent experiments