Role of long non-coding RNAs and RNA-binding proteins in p53 signaling

Division Of Basic Sciences - Nci

Investigators

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Abstract

Our research is focused on investigating the function and mechanism of novel long non-coding (lncRNAs) and RNA-binding proteins (RBPs) in p53 signaling and in colorectal cancer. For our investigations, we perform experiments in cell lines, mice and patient-derived organoids. We reported the discovery and initial functional characterization of two p53-induced lncRNAs that we termed PINCR and PURPL (Chaudhary et al., eLife, 2017; Li et al., Cell Reports, 2017). During the course of these studies, we found that one of the introns in PURPL is not spliced out (intron retention). What is special about this intron? How and why is it not spliced out? These are important questions to address given that intron retention is one of the most prevalent forms of alternative splicing but the least understood. Along these lines, we have developed a novel CRISPR based approach to identify the protein responsible for intron retention in PURPL. Our current efforts are focused on determining the functional consequences of intron retention in PURPL and determining if other lncRNAs are regulated via similar mechanisms. Parallel to our studies on lncRNAs, we are investigating the roles of specific RBPs in p53-mediated tumor suppression and in colorectal cancer. To give an example, we recently discovered that the p53-induced RBP ZMAT3, functions as a splicing factor to control cell growth and proliferation in colorectal cancer cells (Muys et al., Genes Dev, 2021). Current efforts are focused on other p53-regulated RBPs that affect different aspects of RNA metabolism. In a recent publication (Singh et al., MCB, 2025) on a RBP in colorectal cancer, we focused on RNASE1, a ribonuclease known to be secreted by cells and degrade extracellular RNAs. We unexpectedly found that RNASE1, in addition to being secreted, is predominantly localized to the nucleus and functions to inhibit gene expression in human colorectal cancer cells. RNASE1 expression is highly cell type-specific and is restricted to well-differentiated CRC cells where its transcription is activated by the pioneer transcription factor FOXA1. Using CRISPR interference utilizing three independent sgRNAs targeting the RNASE1 locus followed by RNA-seq, we found that upon depletion of RNASE1, most of the differentially expressed RNAs are modestly but significantly upregulated suggesting that RNASE1 predominantly functions to inhibit gene expression. In colorectal cancer patients, RNASE1 is significantly downregulated and high RNASE1 expression is associated with better patient survival, indicating a potential tumor suppressive function. Consistent with this, RNASE1 depletion resulted in increased proliferation and clonogenicity indicating that RNASE1 inhibits the growth of colorectal cancer cells and may function as a tumor suppressor. This initial characterization of endogenous RNASE1 uncovered a function of RNASE1 in inhibition of gene expression and colorectal cancer cell proliferation. We anticipate that our studies on lncRNAs and RBPs will enable us to gain insights in RNA biology and lay the foundation for future translational research.