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PURPL is a p53-induced lncRNA that suppresses basal p53 levels. Here, we investigated PURPL upon p53 activation in liver cancer cells, where it is expressed at significantly higher levels than other cell types. Using isoform sequencing, we discovered novel PURPL transcripts that have a retained intron and/or previously unannotated exons. To determine PURPL function upon p53 activation, we performed transcriptome sequencing (RNA-Seq) after depleting PURPL using CRISPR interference (CRISPRi), followed by Nutlin treatment to induce p53. Strikingly, although loss of PURPL in untreated cells altered the expression of only 7 genes, loss of PURPL resulted in altered expression of ~800 genes upon p53 activation, revealing a context-dependent function of PURPL. Pathway analysis suggested that PURPL is important for fine-tuning the expression of specific genes required for mitosis. Consistent with these results, we observed a significant decrease in the percentage of mitotic cells upon PURPL depletion. Collectively, these data identify novel transcripts from the PURPL locus and suggest that PURPL delicately moderates the expression of mitotic genes in the context of p53 activation to control cell cycle arrest.

Circular RNAs (circRNAs) are a class of noncoding RNAs produced by a noncanonical form of alternative splicing called back-splicing. To investigate a potential role of circRNAs in the p53 pathway, we analyzed RNA sequencing (RNA-seq) data from colorectal cancer cell lines (HCT116, RKO, and SW48) that were untreated or treated with a DNA-damaging agent. Surprisingly, unlike the strong p53-dependent induction of hundreds of p53-induced mRNAs upon DNA damage, only a few circRNAs were upregulated from p53-induced genes. circ-MDM2, an annotated circRNA from the MDM2 locus, was one of the handful of circRNAs that originated from a p53-induced gene. Given the central role of MDM2 in suppressing p53 protein levels and p53 activity, we investigated the function of circ-MDM2. Knocking down circ-MDM2 with small interfering RNAs (siRNAs) that targeted circ-MDM2 did not alter MDM2 mRNA or MDM2 protein levels but resulted in increased basal p53 levels and growth defects in vitro and in vivo. Consistent with these results, transcriptome profiling showed increased expression of several direct p53 targets, reduced retinoblastoma protein (Rb) phosphorylation, and defects in G(1)-S progression upon silencing circ-MDM2. Our results on the initial characterization of circ-MDM2 identify a new player from the MDM2 locus that suppresses p53 levels and cell cycle progression.

The conserved RNA-binding protein Musashi1 (MSI1) has emerged as a key oncogenic factor in numerous solid tumors, including glioblastoma. However, its mechanism of action has not yet been established comprehensively. To identify its target genes comprehensively and determine the main routes by which it influences glioblastoma phenotypes, we conducted individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) experiments. We confirmed that MSI1 has a preference for UAG sequences contained in a particular structural context, especially in 3′ untranslated regions. Although numerous binding sites were also identified in intronic sequences, our RNA transcriptome sequencing analysis does not favor the idea that MSI1 is a major regulator of splicing in glioblastoma cells. MSI1 target mRNAs encode proteins that function in multiple pathways of cell proliferation and cell adhesion. Since these associations indicate potentially new roles for MSI1, we investigated its impact on glioblastoma cell adhesion, morphology, migration, and invasion. These processes are known to underpin the spread and relapse of glioblastoma, in contrast to other tumors where metastasis is the main driver of recurrence and progression.