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- All Subjects: miRNA
- Creators: Mangone, Marco
- Resource Type: Text
- Status: Published
Description
microRNAs (miRNAs) are short ~22nt non-coding RNAs that regulate gene output at the post-transcriptional level. Via targeting of degenerate elements primarily in 3'untranslated regions (3'UTR) of mRNAs, miRNAs can target thousands of varying genes and suppress their protein translation. The precise mechanistic function and bio- logical role of miRNAs is not fully understood and yet it is a major contributor to a pleth- ora of diseases, including neurological disorders, muscular disorders, and cancer. Cer- tain model organisms are valuable in understanding the function of miRNA and there- fore fully understanding the biological significance of miRNA targeting. Here I report a mechanistic analysis of miRNA targeting in C. elegans, and a bioinformatic approach to aid in further investigation of miRNA targeted sequences. A few of the biologically significant mechanisms discussed in this thesis include alternative polyadenylation, RNA binding proteins, components of the miRNA recognition machinery, miRNA secondary structures, and their polymorphisms. This thesis also discusses a novel bioinformatic approach to studying miRNA biology, including computational miRNA target prediction software, and sequence complementarity. This thesis allows a better understanding of miRNA biology and presents an ideal strategy for approaching future research in miRNA targeting.
ContributorsWeigele, Dustin Keith (Author) / Mangone, Marco (Thesis director) / Katchman, Benjamin (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-12
Description
MicroRNAs (miRNAs) are 17-22 nucleotide non-coding RNAs that regulate gene expression by targeting non-complementary elements in the 3’ untranslated regions (3’UTRs) of mRNAs. miRNAs, which form complex networks of interaction that differ by tissue and developmental stage, display conservation in their function across metazoan species. Yet much remains unknown regarding their biogenesis, localization, strand selection, and their absolute abundance due to the difficulty of detecting and amplifying such small molecules. Here, I used an updated HT qPCR-based methodology to follow miRNA expression of 5p and 3p strands for all 190 C. elegans miRNAs described in miRBase throughout all six developmental stages in triplicates (total of 9,708 experiments), and studied their expression levels, tissue localization, and the rules underlying miRNA strand selection. My study validated previous findings and identified novel, conserved patterns of miRNA strand expression throughout C. elegans development, which at times correlate with previously observed developmental phenotypes. Additionally, my results highlighted novel structural principles underlying strand selection, which can be applied to higher metazoans.
Though optimized for use in C. elegans, this method can be easily adapted to other eukaryotic systems, allowing for more scalable quantitative investigation of miRNA biology and/or miRNA diagnostics.
ContributorsMeadows, Dalton Alexander (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Murugan, Vel (Committee member) / Wilson-Rawls, Jeanne (Committee member) / Arizona State University (Publisher)
Created2023
Description
MicroRNAs (miRNAs) are short non-coding RNAs that play key roles during metazoan development, and are frequently misregulated in human disease. MiRNAs regulate gene output by targeting degenerate elements primarily in the 3´ untranslated regions of mRNAs. MiRNAs are often deeply conserved, but have undergone drastic expansions in higher metazoans, leading to families of miRNAs with highly similar sequences. The evolutionary advantage of maintaining multiple copies of duplicated miRNAs is not well understood, nor has the distinct functions of miRNA family members been systematically studied. Furthermore, the unbiased and high-throughput discovery of targets remains a major challenge, yet is required to understand the biological function of a given miRNA.
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.
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.
ContributorsWolter, Justin M (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Kusumi, Kenro (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2016