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Understanding the function of rare non-coding variants represents a significant challenge. Using MapUTR, a screening method, we studied the function of rare 3' UTR variants affecting mRNA abundance post-transcriptionally. Among 17,301 rare gnomAD variants, an average of 24.5% were functional, with 70% in cancer-related genes, many in critical cancer pathways. This observation motivated an interrogation of 11,929 somatic mutations, uncovering 3928 (33%) functional mutations in 155 cancer driver genes. Functional MapUTR variants were enriched in microRNA- or protein-binding sites and may underlie outlier gene expression in tumors. Further, we introduce untranslated tumor mutational burden (uTMB), a metric reflecting the amount of somatic functional MapUTR variants of a tumor and show its potential in predicting patient survival. Through prime editing, we characterized three variants in cancer-relevant genes (MFN2, FOSL2, and IRAK1), demonstrating their cancer-driving potential. Our study elucidates the function of tens of thousands of non-coding variants, nominates non-coding cancer driver mutations, and demonstrates their potential contributions to cancer.

Abstract Understanding the function of rare non-coding variants represents a significant challenge. Using MapUTR, a screening method, we studied the function of rare 3′ UTR variants affecting mRNA abundance post-transcriptionally. Among 17,301 rare gnomAD variants, an average of 24.5% were functional, with 70% in cancer-related genes, many in critical cancer pathways. This observation motivated an interrogation of 11,929 somatic mutations, uncovering 3928 (33%) functional mutations in 155 cancer driver genes. Functional MapUTR variants were enriched in microRNA- or protein-binding sites and may underlie outlier gene expression in tumors. Further, we introduce untranslated tumor mutational burden (uTMB), a metric reflecting the amount of somatic functional MapUTR variants of a tumor and show its potential in predicting patient survival. Through prime editing, we characterized three variants in cancer-relevant genes (MFN2, FOSL2, and IRAK1), demonstrating their cancer-driving potential. Our study elucidates the function of tens of thousands of non-coding variants, nominates non-coding cancer driver mutations, and demonstrates their potential contributions to cancer.

Allele-specific protein-RNA binding is an essential aspect that may reveal functional genetic variants (GVs) mediating post-transcriptional regulation. Recently, genome-wide detection of in vivo binding of RNA-binding proteins is greatly facilitated by the enhanced crosslinking and immunoprecipitation (eCLIP) method. We developed a new computational approach, called BEAPR, to identify allele-specific binding (ASB) events in eCLIP-Seq data. BEAPR takes into account crosslinking-induced sequence propensity and variations between replicated experiments. Using simulated and actual data, we show that BEAPR largely outperforms often-used count analysis methods. Importantly, BEAPR overcomes the inherent overdispersion problem of these methods. Complemented by experimental validations, we demonstrate that the application of BEAPR to ENCODE eCLIP-Seq data of 154 proteins helps to predict functional GVs that alter splicing or mRNA abundance. Moreover, many GVs with ASB patterns have known disease relevance. Overall, BEAPR is an effective method that helps to address the outstanding challenge of functional interpretation of GVs.

RNA editing introduces single nucleotide changes to RNA, thus potentially diversifying gene expression. Recent studies have reported significant changes in RNA editing profiles in disease and development. The functional consequences of these widespread alterations remain elusive because of the unknown function of most RNA editing sites. Here, we carry out a comprehensive analysis of A-to-I editomes in human populations. Surprisingly, we observe highly similar editing profiles across populations despite striking differences in the expression levels of ADAR genes. Striving to explain this discrepancy, we uncover a functional mechanism of A-to-I editing in regulating mRNA abundance. We show that A-to-I editing stabilizes RNA secondary structures and reduces the accessibility of AGO2-miRNA to target sites in mRNAs. The editing-dependent stabilization of mRNAs in turn alters the observed editing levels in the stable RNA repertoire. Our study provides valuable insights into the functional impact of RNA editing in human cells.

Although major genetic networks controlling early liver specification and morphogenesis are known, the mechanisms responsible for postnatal hepatic maturation are poorly understood. Here we employ global analyses of the mouse liver transcriptome to demonstrate that postnatal remodelling of the liver is accompanied by large-scale transcriptional and post-transcriptional transitions that are cell-type-specific and temporally coordinated. Combining detailed expression analyses with gain- and loss-of-function studies, we identify epithelial splicing regulatory protein 2 (ESRP2) as a conserved regulatory factor that controls the neonatal-to-adult switch of ∼20% of splice isoforms in mouse and human hepatocytes. The normal shift in splicing coincides tightly with dramatic postnatal induction of ESRP2 in hepatocytes. We further demonstrate that forced expression of ESRP2 in immature mouse and human hepatocytes is sufficient to drive a reciprocal shift in splicing and causes various physiological abnormalities. These findings define a direct role for ESRP2 in the generation of conserved repertoires of adult splice isoforms that facilitate terminal differentiation and maturation of hepatocytes.

Adenosine deaminases acting on RNA (ADARs) are the primary factors underlying adenosine to inosine (A-to-I) editing in metazoans. Here we report the first global study of ADAR1-RNA interaction in human cells using CLIP-seq. A large number of CLIP sites are observed in Alu repeats, consistent with ADAR1's function in RNA editing. Surprisingly, thousands of other CLIP sites are located in non-Alu regions, revealing functional and biophysical targets of ADAR1 in the regulation of alternative 3' UTR usage and miRNA biogenesis. We observe that binding of ADAR1 to 3' UTRs precludes binding by other factors, causing 3' UTR lengthening. Similarly, ADAR1 interacts with DROSHA and DGCR8 in the nucleus and possibly out-competes DGCR8 in primary miRNA binding, which enhances mature miRNA expression. These functions are dependent on ADAR1's editing activity, at least for a subset of targets. Our study unfolds a broad landscape of the functional roles of ADAR1.

Differential binding of RNA-binding proteins mediated by genetic variants (GVs) can influence posttranscriptional regulation. Here, the authors develop BEAPR, a computational approach to identify allele-specific binding events in eCLIP-Seq data.

RNA A-to-I editing introduces single nucleotide changes to RNA, but its role in cells remains unclear. Here, the authors analyse A-to-I editomes in human samples and find that A-to-I editing stabilizes RNA secondary structures and reduces the accessibility of AGO2-miRNA to target sites in mRNAs.