MicroRNAs (miRNAs) regulate gene output by targeting degenerate elements in mRNAs and have undergone drastic expansions in higher metazoan genomes. The evolutionary advantage of maintaining copies of highly similar miRNAs is not well understood, nor is it clear what unique functions, if any, miRNA family members possess. Here, we study evolutionary patterns of metazoan miRNAs, focusing on the targeting preferences of the let-7 and miR-10 families. These studies reveal hotspots for sequence evolution with implications for targeting and secondary structure. High-throughput screening for functional targets reveals that each miRNA represses sites with distinct features and regulates a large number of genes with cooperative function in regulatory networks. Unexpectedly, given the high degree of similarity, single-nucleotide changes grant miRNA family members with distinct targeting preferences. Together, our data suggest complex functional relationships among miRNA duplications, novel expression patterns, sequence change, and the acquisition of new targets.

Background: Lizards are evolutionarily the most closely related vertebrates to humans that can lose and regrow an entire appendage. Regeneration in lizards involves differential expression of hundreds of genes that regulate wound healing, musculoskeletal development, hormonal response, and embryonic morphogenesis. While microRNAs are able to regulate large groups of genes, their role in lizard regeneration has not been investigated.

Results: MicroRNA sequencing of green anole lizard (Anolis carolinensis) regenerating tail and associated tissues revealed 350 putative novel and 196 known microRNA precursors. Eleven microRNAs were differentially expressed between the regenerating tail tip and base during maximum outgrowth (25 days post autotomy), including miR-133a, miR-133b, and miR-206, which have been reported to regulate regeneration and stem cell proliferation in other model systems. Three putative novel differentially expressed microRNAs were identified in the regenerating tail tip.

Conclusions: Differentially expressed microRNAs were identified in the regenerating lizard tail, including known regulators of stem cell proliferation. The identification of 3 putative novel microRNAs suggests that regulatory networks, either conserved in vertebrates and previously uncharacterized or specific to lizards, are involved in regeneration. These findings suggest that differential regulation of microRNAs may play a role in coordinating the timing and expression of hundreds of genes involved in regeneration.

Mechanism of Notch Signaling: The image depicts a type of cell signaling, in which two animal cells interact and transmit a molecular signal from one to the other. The process results in the production of proteins, which influence the cells as they differentiate, move, and contribute to embryological development. In the membrane of the signaling cell, there is a ligand (represented by a green oval). The ligand functions to activate a change in a receptor molecule. In the receiving cell, there are receptors; in this case, Notch proteins (represented by orange forks). The Notch proteins are embedded in the receiving cell membrane, and they have at least two parts: an intracellular domain (inside the cell) and the receptor (outside the cell). Once the ligand and receptor bind to each other, a protease (represented by the dark red triangle) can sever the intracellular domain from the rest of the Notch receptor. Inside the nucleus of the receiving cell (represented by the gray area) are the cellês DNA (represented by the multi-colored helices) and its transcription factors (blue rectangles). Transcription factors are proteins that bind to DNA to regulate transcription, the first step in gene expression, which eventually yields proteins or other products. Initially, repressor proteins (represented by a red irregular hexagon) prevent transcription factors from allowing transcription. When the severed Notch receptor intracellular domain reaches the nucleus, it displaces the repressor. The transcription factor can then signal for transcription to occur. 1) There is a Notch receptor protein in the membrane of a receiving cell, and a ligand for this receptor (for example, Delta) in the membrane of the signaling cell. When the ligand binds to the receptor, the intracellular domain of the receptor changes shape. 2) Inside the receiving cell, there are proteases. Once the intracellular domain of the receptor changes shape, the protease can bind to it and shear the intracellular domain away from the rest of the receptor molecule. 3) The severed intracellular domain is shuttled to the receiving cell nucleus. Here, the intracellular domain displaces a repressor protein. This allows a transcription factor to initiate DNA transcription. During transcription, DNA is used as a template to create RNA. Following transcription, the process of translation occurs, which uses RNA as a template to create proteins. These proteins influence the behavior, fate, and differentiation of cells, which contribute to normal embryonic development

'On the Permanent Life of Tissues outside of the Organism' reports Alexis Carrel's 1912 experiments on the maintenance of tissue in culture media. At the time, Carrel was a French surgeon and biologist working at the Rockefeller Institute in New York City. In his paper, Carrel reported that he had successfully maintained tissue cultures, which derived from connective tissues of developing chicks and other tissue sources, by serially culturing them. Among all the tissue cultures Carrel reported, one was maintained for more than two months, whereas previous efforts had only been able to keep tissues in vitro for three to fifteen days. Carrel’s experiments contributed to the development of long-term tissue culture techniques, which were useful in the study of embryology and eventually became instrumental in stem cell research. Despite later evidence to the contrary, Carrel believed that as long as the tissue culture method was accurately applied, tissues kept outside of the organisms should be able to divide indefinitely and have permanent life.

Christiane Nusslein-Volhard studied how genes control embryonic development in flies and in fish in Europe during the twentieth and twenty-first centuries. In the 1970s, Nusslein-Volhard focused her career on studying the genetic control of development in the fruit fly Drosophila melanogaster. In 1988, Nusslein-Volhard identified the first described morphogen, a protein coded by the gene bicoid in flies. In 1995, along with Eric F. Wieschaus and Edward B. Lewis, she received the Nobel Prize in Physiology or Medicine for the discovery of genes that establish the body plan and segmentation in Drosophila. Nusslein-Volhard also investigated the genetic control of embryonic development to zebrafish, further generalizing her findings and helping establishing zebrafish as a model organism for studies of vertebrate development.

In 1969, Roy J. Britten and Eric H. Davidson published Gene Regulation for Higher Cells: A Theory, in Science. A Theory proposes a minimal model of gene regulation, in which various types of genes interact to control the differentiation of cells through differential gene expression. Britten worked at the Carnegie Institute of Washington in Washington, D.C., while Davidson worked at the California Institute of Technology in Pasadena, California. Their paper was an early theoretical and mechanistic description of gene regulation in higher organisms.

Bicoid is the protein product of a maternal-effect gene unique to flies of the genus Drosophila . In 1988 Christiane Nüsslein-Volhard identified bicoid as the first known morphogen . A morphogen is a molecule that determines the fate and phenotype of a group of cells through a concentration gradient across that developing region. The bicoid gradient, which extends across the anterior-posterior axis of Drosophila embryos, organizes the head and thorax.

Mesenchyme is a type of animal tissue comprised of loose cells embedded in a mesh of proteins and fluid, called the extracellular matrix. The loose, fluid nature of mesenchyme allows its cells to migrate easily and play a crucial role in the origin and development of morphological structures during the embryonic and fetal stages of animal life. Mesenchyme directly gives rise to most of the body's connective tissues, from bones and cartilage to the lymphatic and circulatory systems. Furthermore, the interactions between mesenchyme and another tissue type, epithelium, help to form nearly every organ in the body.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene output at the post-transcriptional level by targeting degenerate elements primarily in 3′untranslated regions (3′UTRs) of mRNAs. Individual miRNAs can regulate networks of hundreds of genes, yet for the majority of miRNAs few, if any, targets are known. Misexpression of miRNAs is also a major contributor to cancer progression, thus there is a critical need to validate miRNA targets in high-throughput to understand miRNAs' contribution to tumorigenesis. Here we introduce a novel high-throughput assay to detect miRNA targets in 3′UTRs, called Luminescent Identification of Functional Elements in 3′UTRs (3′LIFE). We demonstrate the feasibility of 3′LIFE using a data set of 275 human 3′UTRs and two cancer-relevant miRNAs, let-7c and miR-10b, and compare our results to alternative methods to detect miRNA targets throughout the genome. We identify a large number of novel gene targets for these miRNAs, with only 32% of hits being bioinformatically predicted and 27% directed by non-canonical interactions. Functional analysis of target genes reveals consistent roles for each miRNA as either a tumor suppressor (let-7c) or oncogenic miRNA (miR-10b), and preferentially target multiple genes within regulatory networks, suggesting 3′LIFE is a rapid and sensitive method to detect miRNA targets in high-throughput.

Background: 3′untranslated regions (3′UTRs) are poorly understood portions of eukaryotic mRNAs essential for post-transcriptional gene regulation. Sequence elements in 3′UTRs can be target sites for regulatory molecules such as RNA binding proteins and microRNAs (miRNAs), and these interactions can exert significant control on gene networks. However, many such interactions remain uncharacterized due to a lack of high-throughput (HT) tools to study 3′UTR biology. HT cloning efforts such as the human ORFeome exemplify the potential benefits of genomic repositories for studying human disease, especially in relation to the discovery of biomarkers and targets for therapeutic agents. Currently there are no publicly available human 3′UTR libraries. To address this we have prepared the first version of the human 3′UTRome (h3′UTRome v1) library. The h3′UTRome is produced to a single high quality standard using the same recombinational cloning technology used for the human ORFeome, enabling universal operating methods and high throughput experimentation. The library is thoroughly sequenced and annotated with simple online access to information, and made publicly available through gene repositories at low cost to all scientists with minimal restriction.

Results: The first release of the h3′UTRome library comprises 1,461 human 3′UTRs cloned into Gateway® entry vectors, ready for downstream analyses. It contains 3′UTRs for 985 transcription factors, 156 kinases, 171 RNA binding proteins, and 186 other genes involved in gene regulation and in disease. We demonstrate the feasibility of the h3′UTRome library by screening a panel of 87 3′UTRs for targeting by two miRNAs: let-7c, which is implicated in tumorigenesis, and miR-221, which is implicated in atherosclerosis and heart disease. The panel is enriched with genes involved in the RAS signaling pathway, putative novel targets for the two miRNAs, as well as genes implicated in tumorigenesis and heart disease.

Conclusions: The h3′UTRome v1 library is a modular resource that can be utilized for high-throughput screens to identify regulatory interactions between trans-acting factors and 3′UTRs, Importantly, the library can be customized based on the specifications of the researcher, allowing the systematic study of human 3′UTR biology.