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mRNA expression dynamics promote and maintain the identity of somatic tissues in living organisms; however, their impact in post-transcriptional gene regulation in these processes is not fully understood. Here, we applied the PAT-Seq approach to systematically isolate, sequence, and map tissue-specific mRNA from five highly studied Caenorhabditis elegans somatic tissues: GABAergic and NMDA neurons, arcade and intestinal valve cells, seam cells, and hypodermal tissues, and studied their mRNA expression dynamics. The integration of these datasets with previously profiled transcriptomes of intestine, pharynx, and body muscle tissues, precisely assigns tissue-specific expression dynamics for 60% of all annotated C. elegans protein-coding genes, providing an important resource for the scientific community. The mapping of 15,956 unique high-quality tissue-specific polyA sites in all eight somatic tissues reveals extensive tissue-specific 3'untranslated region (3'UTR) isoform switching through alternative polyadenylation (APA) . Almost all ubiquitously transcribed genes use APA and harbor miRNA targets in their 3'UTRs, which are commonly lost in a tissue-specific manner, suggesting widespread usage of post-transcriptional gene regulation modulated through APA to fine tune tissue-specific protein expression. Within this pool, the human disease gene C. elegans orthologs rack-1 and tct-1 use APA to switch to shorter 3'UTR isoforms in order to evade miRNA regulation in the body muscle tissue, resulting in increased protein expression needed for proper body muscle function. Our results highlight a major positive regulatory role for APA, allowing genes to counteract miRNA regulation on a tissue-specific basis.

BACKGROUND:Tissue-specific RNA plasticity broadly impacts the development, tissue identity and adaptability of all organisms, but changes in composition, expression levels and its impact on gene regulation in different somatic tissues are largely unknown. Here we developed a new method, polyA-tagging and sequencing (PAT-Seq) to isolate high-quality tissue-specific mRNA from Caenorhabditis elegans intestine, pharynx and body muscle tissues and study changes in their tissue-specific transcriptomes and 3'UTRomes. RESULTS:We have identified thousands of novel genes and isoforms differentially expressed between these three tissues. The intestine transcriptome is expansive, expressing over 30% of C. elegans mRNAs, while muscle transcriptomes are smaller but contain characteristic unique gene signatures. Active promoter regions in all three tissues reveal both known and novel enriched tissue-specific elements, along with putative transcription factors, suggesting novel tissue-specific modes of transcription initiation. We have precisely mapped approximately 20,000 tissue-specific polyadenylation sites and discovered that about 30% of transcripts in somatic cells use alternative polyadenylation in a tissue-specific manner, with their 3'UTR isoforms significantly enriched with microRNA targets. CONCLUSIONS:For the first time, PAT-Seq allowed us to directly study tissue specific gene expression changes in an in vivo setting and compare these changes between three somatic tissues from the same organism at single-base resolution within the same experiment. We pinpoint precise tissue-specific transcriptome rearrangements and for the first time link tissue-specific alternative polyadenylation to miRNA regulation, suggesting novel and unexplored tissue-specific post-transcriptional regulatory networks in somatic cells.

Three-prime untranslated regions (3'UTRs) of metazoan messenger RNAs (mRNAs) contain numerous regulatory elements, yet remain largely uncharacterized. Using polyA capture, 3' rapid amplification of complementary DNA (cDNA) ends, full-length cDNAs, and RNA-seq, we defined approximately 26,000 distinct 3'UTRs in Caenorhabditis elegans for approximately 85% of the 18,328 experimentally supported protein-coding genes and revised approximately 40% of gene models. Alternative 3'UTR isoforms are frequent, often differentially expressed during development. Average 3'UTR length decreases with animal age. Surprisingly, no polyadenylation signal (PAS) was detected for 13% of polyadenylation sites, predominantly among shorter alternative isoforms. Trans-spliced (versus non-trans-spliced) mRNAs possess longer 3'UTRs and frequently contain no PAS or variant PAS. We identified conserved 3'UTR motifs, isoform-specific predicted microRNA target sites, and polyadenylation of most histone genes. Our data reveal a rich complexity of 3'UTRs, both genome-wide and throughout development.

Three-prime untranslated regions (3'UTRs) are widely recognized as important post-transcriptional regulatory regions of mRNAs. RNA-binding proteins and small non-coding RNAs such as microRNAs (miRNAs) bind to functional elements within 3'UTRs to influence mRNA stability, translation and localization. These interactions play many important roles in development, metabolism and disease. However, even in the most well-annotated metazoan genomes, 3'UTRs and their functional elements are not well defined. Comprehensive and accurate genome-wide annotation of 3'UTRs and their functional elements is thus critical. We have developed an open-access database, available at http://www.UTRome.org, to provide a rich and comprehensive resource for 3'UTR biology in the well-characterized, experimentally tractable model system Caenorhabditis elegans. UTRome.org combines data from public repositories and a large-scale effort we are undertaking to characterize 3'UTRs and their functional elements in C. elegans, including 3'UTR sequences, graphical displays, predicted and validated functional elements, secondary structure predictions and detailed data from our cloning pipeline. UTRome.org will grow substantially over time to encompass individual 3'UTR isoforms for the majority of genes, new and revised functional elements, and in vivo data on 3'UTR function as they become available. The UTRome database thus represents a powerful tool to better understand the biology of 3'UTRs.