In the face of random perturbations in environment as well as genetic mutations, complex organisms manage to maintain remarkably robust developmental stability. Despite originating from a single cell and sharing a common genome, tissue-based organisms contain many different cellular phenotypes. The robustness of the developmental programs of these organisms depends on their underlying gene regulatory network (GRN), which is composed of a diverse set of mechanisms that help the cell achieve a desired state and stay there. One curiosity of tissue-based organisms is their near-universal dependence on non-coding RNAs, of which the largest class is the 18-22nt long microRNAs (miRNAs). These short RNAs typically, but not always, repress the translation of messenger RNAs (mRNAs) into functional proteins in a sequence-specific manner. Nonetheless, identifying the relationship between tissue-based organism development and miRNAs has been challenging. Within a single model organism, a large number of synthesis and functional pathways contribute to this overall effect. Furthermore, despite their overall dependence on miRNAs, it has been demonstrated that only a small subset of the total set of DNA-encoded miRNAs (the miRNAome) is required for proper development, and the loss of individual miRNAs often causes no change in phenotype. This dissertation aims to postulate a "fundamental law of miRNAs" in the development of these organisms. The complexities of miRNA-mediated gene regulation can often obscure their commonalities; by using a common abstraction of GRNs and gene expression, my aim is to show that miRNA-mediated regulation is innately tied to the notion of stem cells and cell differentiation. Using a multiple different models of gene expression and cell differentiation, I show how the specific type of regulation imparted by miRNAs can help create a stable, undifferentiated state and control the duration of that state. This role for miRNAs is supported independently by each model and agrees with experimentally-observed data.

This detailed volume provides a collection of protocols for the study of miRNA functions in plants. Beginning with coverage of miRNA function, biogenesis, activity, and evolution in plants, the book continues by guiding readers through methods on the identification and detection of plant miRNAs, bioinformatic analyses, and strategies for functional analyses of miRNAs. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Plant MicroRNAs: Method and Protocols aims to ensure successful results in the further study of this vital area of plant science.

This cutting-edge text will be a valuable resource for scientists and aspiring graduate students interested in the intersection of RNAi, miRNA, and stem cell molecular biology and exciting areas of research such as regenerative medicine and cancer.

Given this pervasiveness and importance of miRNA-mediated gene regulation, it should come as little surprise that miRNAs themselves are also highly regulated. However, the recent explosion of knowledge on this topic has been remarkable, providing a primary motivation for publication of this book. As miRNAs are transcribed by RNA polymerase II, the enzyme that also generates mRNAs, it was perhaps not unexpected that miRNA transcription would be subject to regulation, and we have willfully mitted this aspect from this monograph. However, what has been unexpected is the extent of post-transcriptional regulation of miRNAs that is illustrated in this book.

New Findings Revolutionize Concepts of Gene FunctionEndogenous small RNAs have been found in various organisms, including humans, mice, flies, worms, fungi, and bacteria. Furthermore, it's been shown that microRNAs acting as cellular rheostats have the ability to modulate gene expression. In higher eukaryotes, microRNAs may regulate as much as 50 p

MicroRNAs have recently emerged as key regulators of gene expression during development and are frequently misexpressed in human disease states, in particular cancer. These 22-nucleotide-long transcripts act to promote or repress cell proliferation, migration and apoptosis during development, all of which are processes that go awry in cancer. Thus, microRNAs have the ability to behave like oncogenes or tumor suppressors. In addition, their small size and molecular properties make them amenable as targets and therapeutics in cancer treatment. This book goes into detail on how microRNAs represent a paradigm shift in thinking about gene regulation during development and disease, and provide the oncologist with a potentially powerful new battery of agents to diagnose and treat cancer.

"Post-transcriptional gene regulation plays an essential role in controlling animal development and diseases. The 3ʹ untranslated region (3ʹUTR) of messenger RNAs (mRNAs) mainly defines how this regulation is enacted. microRNAs (miRNAs) are short RNAs of approximately 21 nt and along with their associated miRNA-induced silencing complex (miRISC), bind to 3ʹUTRs and direct mRNA translation inhibition, deadenylation, and decay. Many RNA-binding proteins (RBPs) mediate similar post-transcriptional gene regulation. The depth of interplay between 3ʹUTRs, miRNAs, and RBPs remains misunderstood and often underappreciated. Cooperation between miRNA binding sites is commonplace, but its importance in vivo remains debated. We have combined biochemical and genetic tools to study cooperative interactions between trans-acting factors in the nematode Caenorhabditis elegans.Employing a cell-free extract derived from C. elegans embryos, we unmasked multiple layers of cooperativity in miRNA-mediated gene silencing. First, we found that miRISC bound to 3ʹUTRs facilitate the recruitment of miRNAs to adjacent binding sites. Furthermore, we demonstrated that silencing is carried out through the cooperative recruitment of the Ccr4-Not deadenylase complex. Using a structural model of cooperating miRISCs, we identified allosteric determinants of cooperative miRNA-mediated silencing that are required for miRNA function in vivo. miRNA-mediated silencing is modulated by the length and structure of 3ʹUTRs. We implicated the poly(A) binding proteins PAB-1 and PAB-2 in this modulation and showed that they promote deadenylation and are required for miRNA function in vivo. Finally, we investigated and demonstrated cooperation between the miRISC and NHL-2, a poly(U) binding protein. A proteomic survey of NHL-2 revealed extensive interactions with the miRNA and deadenylation machinery and we found that NHL-2 binding sites strongly enhances miRNA-directed mRNA deadenylation. Our results delineate multiple cooperative mechanisms at play in 3ʹUTRs and further support the consideration of target site cooperation as a fundamental characteristic of post-transcriptional gene regulation." --