Recent transcriptome data from yeast, worm, plants, and humans has shown that alternative polyadenylation (APA), a mechanism that enables expression of multiple 3′UTR isoforms for the same gene, is widespread in eukaryotic organisms. It is still poorly understood why metazoans require multiple 3′UTRs for the same gene, but accumulating evidence suggests that APA is largely regulated at a tissue-specific level. APA may direct combinatorial variation between cis-elements and microRNAs, perhaps to regulate gene expression in a tissue-specific manner. Apart from a few single gene anecdotes, this idea has not been systematically explored.
This dissertation research employs a systems biology approach to study the somatic tissue dynamics of APA and its impact on microRNA targeting networks in the small nematode C. elegans. In the first aim, tools were developed and applied to isolate and sequence mRNA from worm intestine and muscle tissues, which revealed pervasive tissue-specific APA correlated with microRNA regulation. The second aim provides genetic evidence that two worm genes use APA to escape repression by microRNAs in the body muscle. Finally, in aim three, mRNA from five additional somatic worm tissues was sequenced and their 3′ends mapped, allowing for an integrative study of APA and microRNA targeting dynamics in worms. Together, this work provides evidence that APA is a pervasive mechanism operating in somatic tissues of C. elegans with the potential to significantly rearrange their microRNA regulatory networks and precisely dose their gene expression.
I hypothesize that duplication events grant miRNA families with enhanced regulatory capabilities, specifically through distinct targeting preferences by family members. This has relevance for our understanding of vertebrate evolution, as well disease detection and personalized medicine. To test this hypothesis, I apply a conjunction of bioinformatic and experimental approaches, and design a novel high-throughput screening platform to identify human miRNA targets. Combined with conventional approaches, this tool allows systematic testing for functional targets of human miRNAs, and the identification of novel target genes on an unprecedented scale.
In this dissertation, I explore evolutionary signatures of 62 deeply conserved metazoan miRNA families, as well as the targeting preferences for several human miRNAs. I find that constraints on miRNA processing impact sequence evolution, creating evolutionary hotspots within families that guide distinct target preferences. I apply our novel screening platform to two cancer-relevant miRNAs, and identify hundreds of previously undescribed targets. I also analyze critical features of functional miRNA target sites, finding that each miRNA recognizes surprisingly distinct features of targets. To further explore the functional distinction between family members, I analyze miRNA expression patterns in multiple contexts, including mouse embryogenesis, RNA-seq data from human tissues, and cancer cell lines. Together, my results inform a model that describes the evolution of metazoan miRNAs, and suggests that highly similar miRNA family members possess distinct functions. These findings broaden our understanding of miRNA function in vertebrate evolution and development, and how their misexpression contributes to human disease.
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.
Biomarkers encompass a wide range of different measurable indicators, representing a tangible link to physiological changes occurring within the body. Accessibility, sensitivity, and specificity are significant factors in biomarker suitability. New biomarkers continue to be discovered, and questions over appropriate selection and assessment of their usefulness remain. If traditional markers of inflammation are not sufficiently robust in their specificity, then perhaps alternative means of detection may provide more information. Epigenetic drift (epigenetic modifications as they occur as a direct function with age), and its ancillary elements, including platelets, secreted microvesicles (MVs), and microRNA (miRNA), may hold enormous predictive potential. The majority of epigenetic drift observed in blood is independent of variations in blood cell composition, addressing concerns affecting traditional blood-based biomarker efficacy. MVs are found in plasma and other biological fluids in healthy individuals. Altered MV/miRNA profiles may also be found in individuals with various diseases. Platelets are also highly reflective of physiological and lifestyle changes, making them extremely sensitive biomarkers of human health. Platelets release increased levels of MVs in response to various stimuli and under a plethora of disease states, which demonstrate a functional effect on other cell types.