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.

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.

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