Genetics and Genomics of Alternative Polyadenylation and miRNA Regulation in C. e - Renewal - 1

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Abstract 3’ Untranslated Regions (3’UTR) are mRNA ends located between the STOP codon and the polyA tail. They contain numerous regulatory elements that are poorly characterized, making them a core target for post- transcriptional regulation of gene expression by miRNAs and RNA binding proteins. MiRNAs are small regulatory RNAs that bind 3’UTRs and repress mRNA translation. Our group and others have shown that alternative polyadenylation (APA), an elusive process observed in metazoans that gives rise to genes with various 3’UTR isoforms, is widespread. While APA is frequently present in normal states and in virtually all diseases, its biological role in cells and tissues is a mystery. Genes with longer 3’UTRs have more potential regulatory sites, thus APA may allow genes to alter their 3’UTR length and escape the repression by miRNAs and RNA binding proteins. Since APA is abundant this could explain why there is not a direct correlation between cellular transcriptomes and proteomes. Understanding the global mechanics of 3’end formation, APA function, and miRNA targeting would advance research on post-transcriptional modifications. Metazoans possess hundreds of miRNAs and thousands of 3’UTR isoforms, most of which are expressed and function in different tissues and developmental contexts. We know little about how these networks operate and communicate with each other, despite their critical role in controlling gene expression. We need to gain new data and insights, and develop a high-resolution miRNAs/3’UTRs Interactome by acquiring and fusing tissue- specific miRNA localization data, miRNA target data, and 3’UTRs isoform data. Our Interactome will provide the global basis of post-transcriptional gene regulation function in vivo, elucidating its role in both normal and disease states in metazoans. In our first R01 grant we studied tissue-specific APA events and miRNA networks in the nematode C. elegans, to define and probe the worm miRNAs/3’UTRs Interactome. We identified 3’UTR isoforms in eight C. elegans somatic tissues, acquired miRNA expression data for the three largest and disease-relevant C. elegans worm tissues, developed and implemented unbiased methods to map miRNA targets in high throughput, and initiated mechanistic studies of APA selection in genes identified in our studies. For this new submission we will, (1) complete the worm 3’UTRome by including new tissue-specific 3’UTR isoforms, (2) identify tissue-specific miRNA populations in eight worm somatic tissues and, (3) perform mechanistic studies to determine tissue-specific PAS choice in the context of APA. Our curated 3’UTRome will be the first database to provide a complete metazoan 3’UTRome mapped at a single base resolution. Investigating three global hypotheses of PAS choice can uncover global post-transcriptional gene regulatory mechanisms implicated in tissue development and disease, and therefore of high impact. We believe that the impact of these new studies is high since will provide important results toward a global understanding the biology of post-transcriptional gene regulation in metazoans. 1