PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

In tumors with WT p53, alternate mechanisms of p53 inactivation are reported. Here, we have identified a long noncoding RNA, PITAR ( p 53 I nactivating T RIM28 a ssociated R NA), as an inhibitor of p53. PITAR is an oncogenic Cancer/testis lncRNA and is highly expressed in glioblastoma (GBM) and glioma stem-like cells (GSC). We establish that TRIM28 mRNA, which encodes a p53-specific E3 ubiquitin ligase, is a direct target of PITAR. PITAR interaction with TRIM28 RNA stabilized TRIM28 mRNA, which resulted in increased TRIM28 protein levels and reduced p53 steady-state levels due to enhanced p53 ubiquitination. DNA damage activated PITAR, in addition to p53, in a p53-independent manner, thus creating an incoherent feedforward loop to inhibit the DNA damage response by p53. While PITAR silencing inhibited the growth of WT p53 containing GSCs in vitro and reduced glioma tumor growth in vivo, its overexpression enhanced the tumor growth in a TRIM28-dependent manner and promoted resistance to ..., The targets of LncRNA were identified using ChIRP-RNA sequencing, which was described earlier (Chu, Quinn and Chang, 2012). Antisense DNA probes for PITAR were designed using the Stellaris Probe Designer tool. Probes were labeled with Biotin-TEG at the 3′ ends. U87 cells were crosslinked with 1% glutaraldehyde for 10 min at 37 °C and then quenched with 0.125 M glycine buffer for 5 min. U87 cells were lysed in lysis buffer (50 mM Tris, pH 7.0, 10 mM EDTA, 1% SDS, DTT, PMSF, protease inhibitor, and RNase inhibitor) on ice for 30 min, and genomes were sonicated three times into fragments 300–500 bp in length. Chromatins were diluted twice the volume of hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris, pH 7.0, 1 mM EDTA, 15% formamide, DTT, PMSF, protease inhibitor and RNase inhibitor). Biotin-TEG labeled probes (odd, even, and LacZ) were added, and mixtures were rotated at 37 °C for four hours. Streptavidin-magnetic C1 beads were blocked with 500 ng/µl yeast total RNA and 1 mg/ml BSA ..., , # Data from: PITAR, a DNA damage-inducible Cancer/Testis long noncoding RNA, inactivates p53 by binding and stabilizing TRIM28 mRNA

[https://doi.org/10.5061/dryad.3j9kd51t7](https://doi.org/10.5061/dryad.3j9kd51t7)

We have submitted our normalized gene abundance data (**ChIRP_RNA-Seq_Data.xlsx and siFAM95B1_RNA-Seq_Data.xlsx**). The raw reads were quality-analyzed, and upon satisfactory assessment, Kallisto was used with default parameters to quantitate and obtain TPM-normalized gene abundance. The fold change was brought using Deseq2.

**Descriptions:**

**ChIRP_RNA-Seq_Data**

Chromatin Isolation by RNA Purification (ChIRP), performed using biotinylated FAM95B1 (PITAR) specific antisense probes labeled with Biotin-TEG at the 3′ ends. We incubated biotin probes with U87 cell lysates and then used Streptavidin C1 magnetic beads for RNA purification. The beads were then treated with RNA elution buffer to release the RNA, which was subsequently subjected to total RNA sequencing. Here we...