Acinus regulates intron retention in the DFFA pre-mRNA, which encodes an apoptotic regulator, as well as of pre-mRNAs encoding RNA processing factors and cell cycle regulators. (A) Structure of DFFA transcripts. The transcription start site and the direction of transcription are indicated by an arrow. The CLIP+ intron (retained in transcript 2) is indicated in red. Arrows underneath indicate primers used for RT-qPCR analysis to detect specifically transcript 2 or both transcripts t1 and t2. (B) Ratio of DFFA transcript 2 compared to total DFFA transcript. The quantification of transcript 2 and total transcript was done by RT-qPCR using specific primers. The quantification was done on four replicates for control cells and five replicates for knockdown 1 and 2 of Acinus. (*) P-value below 0.05 as calculated using the Mann-Whitney test. (C) Graph showing selected enriched GO terms for genes bound by Acinus (4416 genes). This analysis was done using DAVID functional annotation tool and ranked using the Benjamini corrected P-value. (D) Graph showing selected enriched GO term for genes that show binding of Acinus in introns (2668 genes). This analysis was done using DAVID functional annotation tool, as described above. (E) Cell cycle profile of control and Acinus-depleted HeLa cells following release from a G1 arrest at 0, 6, 9, and 12 h. After siRNA treatment, cells were arrested in G1 using 2 mM mimosime treatment for 20 h. After release, cells were collected at different time points. The cell cycle profile was obtained by propidium iodide staining and flow cytometry.
