# LINC01021 Attenuates Expression and Affects Alternative Splicing of a Subset of p53-Regulated Genes

## TL;DR

This study shows that the long noncoding RNA LINC01021 influences how p53 regulates gene expression and splicing, affecting tumor suppression and chemotherapy response in colorectal cancer.

## Contribution

The paper reveals that LINC01021 modulates p53-regulated gene expression and splicing, beyond its known role in p53 feedback regulation.

## Key findings

- Loss of LINC01021 increases the number of p53-regulated mRNAs and enhances the induction of genes like NOXA and FAS.
- LINC01021 affects alternative splicing of genes such as ARHGAP12, HSF2, and LYN.
- RNA binding proteins involved in splicing interact with LINC01021, suggesting a role in RNA processing.

## Abstract

Growing evidence indicates that p53-induced long noncoding (lnc) RNAs constitute an elaborate regulatory network that mediates and/or modulates p53 function and, thus, tumor suppression. p53-induced LINC01021 has been suggested to represent a negative feedback regulator of p53 protein stability under non-stress conditions. Furthermore, the loss of LINC01021 in p53-proficient colorectal cancer (CRC) cell lines results in increased sensitivity to DNA-damaging chemotherapeutics. In order to analyze whether LINC01021 affects the transcriptional program of p53 independently from the direct feedback regulation of p53, we studied the effect of CRISPR/Cas9-mediated abrogation of p53-induced LINC01021 transcription on genome-wide RNA expression changes after the activation of ectopic p53 in CRC cells by RNA-Seq analyses. Our results demonstrate diverse regulatory effects of LINC01021 on a subset of p53-regulated genes either via attenuated expression or altered transcript isoform usage. Taken together, our study provides a comprehensive framework and resource for further analyses of LINC01021 function downstream of p53.

Background: Loss of the p53-inducible LINC01021 in p53-proficient CRC cell lines results in increased sensitivity to DNA-damaging chemotherapeutics. Here, we comprehensively analyze how LINC01021 affects the p53-induced transcriptional program. Methods: Using a CRISPR/Cas9-approach, we deleted the p53 binding site in the LINC01021 promoter of SW480 colorectal cancer cells and subjected them to RNA-Seq analysis after the activation of ectopic p53. RNA affinity purification followed by mass spectrometry was used to identify proteins associated with LINC01021. Results: Loss of the p53-inducibility of LINC01021 resulted in an ~1.8-fold increase in the number of significantly regulated mRNAs compared to LINC01021 wild-type cells after ectopic activation of p53. A subset of direct p53 target genes, such as NOXA and FAS, displayed significantly stronger induction when the p53-inducibility of LINC01021 was abrogated. Loss of the p53-inducibility of LINC01021 resulted in alternative splicing of a small number of mRNAs, such as ARHGAP12, HSF2, and LYN. Several RNA binding proteins involved in pre-mRNA splicing were identified as interaction partners of LINC01021 by mass spectrometry. Conclusions: Our results suggest that LINC01021 may restrict the extent and strength of p53-mediated transcriptional changes via context-dependent regulation of the expression and splicing of a subset of p53-regulated genes.