BMP Signaling Goes Posttranscriptional in a microRNA Sort of Way

Aberrant microRNA (miRNA) expression correlates with human diseases such as cardiac disorders and cancer. Treatment of such disorders using miRNA-targeted therapeutics requires a thorough understanding of miRNA regulation in vivo. A recent paper in Nature by Davis et al. expands our understanding of miRNA biogenesis and maturation, elucidating a mechanism by which extracellular signaling directs cell differentiation via posttranscriptional regulation of miRNA expression

Identifying a New Gene Required for microRNA-mediated Gene Silencing in \u3cem\u3eDrosophila melanogaster\u3c/em\u3e

Mature microRNAs (miRNA) are ~22 nucleotide long single-stranded ribonucleic acids essential for gene silencing. Silencing occurs when miRNAs are processed via endonucleolytic cleavage and subsequently associate with the miRNA-induced silencing complex (miRISC). miRISC binds via complementary base pairing to target mRNAs, and target mRNAs are silenced by either mRNA degradation, translational block, or both. Knowledge of all genes required for silencing is incomplete. We aim to determine the molecular mechanism of silencing by identifying and characterizing genes required for silencing. A forward genetic screen was performed using EMS mutagenesis of Drosophila melanogaster to generate mutant lines with disrupted gene silencing as visualized by a GFP-based fluorescent reporter of silencing. Locations of EMS-induced mutations are being mapped by determination of recombination frequencies between these mutations and molecularly defined P-element insertions. Preliminary recombination mapping reveals that our mutation of interest (I1-5) is found within a discrete region of the genome on chromosome 3R. A new fly line has also been generated to assist with this preliminary recombination mapping. Future deficiency mapping and complementation tests combining the mutation and alleles of candidate genes will reveal the location of our mutation, and lead us to identify a gene required for micro-RNA mediated gene silencing

A microRNA Imparts Robustness against Environmental Fluctuation during Development

The microRNA miR-7 is perfectly conserved from annelids to humans, and yet some of the genes that it regulates in Drosophila are not regulated in mammals. We have explored the role of lineage restricted targets, using Drosophila , in order to better understand the evolutionary significance of microRNA-target relationships. From studies of two well characterized developmental regulatory networks, we find that miR-7 functions in several interlocking feedback and feedforward loops, and propose that its role in these networks is to buffer them against perturbation. To directly demonstrate this function for miR-7, we subjected the networks to temperature fluctuation and found that miR-7 is essential for the maintenance of regulatory stability under conditions of environmental flux. We suggest that some conserved microRNAs like miR-7 may enter into novel genetic relationships to buffer developmental programs against variation and impart robustness to diverse regulatory networks

Design of Peptide Inhibitors That Bind the bZIP Domain of Epstein–Barr Virus Protein BZLF1

Designing proteins or peptides that bind native protein targets can aid the development of novel reagents and/or therapeutics. Rational design also tests our understanding of the principles underlying protein recognition. This article describes several strategies used to design peptides that bind to the basic region leucine zipper (bZIP) domain of the viral transcription factor BZLF1, which is encoded by the Epstein–Barr virus. BZLF1 regulates the transition of the Epstein–Barr virus from a latent state to a lytic state. It shares some properties in common with the more studied human bZIP transcription factors, but also includes novel structural elements that pose interesting challenges to inhibitor design. In designing peptides that bind to BZLF1 by forming a coiled-coil structure, we considered both affinity for BZLF1 and undesired self-association, which can weaken the effectiveness of an inhibitor. Several designed peptides exhibited different degrees of target-binding affinity and self-association. Rationally engineered molecules were more potent inhibitors of DNA binding than a control peptide corresponding to the native BZLF1 dimerization region itself. The most potent inhibitors included both positive and negative design elements and exploited interaction with the coiled-coil and basic DNA-binding regions of BZLF1.David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship)National Institutes of Health (U.S.) (Award GM067681)National Science Foundation (U.S.) (Award 0821391

A Role for Actin, Cdc1p, and Myo2p in the Inheritance of Late Golgi Elements in \u3cem\u3eSaccharomyces cerevisiae\u3c/em\u3e

In Saccharomyces cerevisiae, Golgi elements are present in the bud very early in the cell cycle. We have analyzed this Golgi inheritance process using fluorescence microscopy and genetics. In rapidly growing cells, late Golgi elements show an actin-dependent concentration at sites of polarized growth. Late Golgi elements are apparently transported into the bud along actin cables and are also retained in the bud by a mechanism that may involve actin. A visual screen for mutants defective in the inheritance of late Golgi elements yielded multiple alleles of CDC1. Mutations in CDC1 severely depolarize the actin cytoskeleton, and these mutations prevent late Golgi elements from being retained in the bud. The efficient localization of late Golgi elements to the bud requires the type V myosin Myo2p, further suggesting that actin plays a role in Golgi inheritance. Surprisingly, early and late Golgi elements are inherited by different pathways, with early Golgi elements localizing to the bud in a Cdc1p- and Myo2p-independent manner. We propose that early Golgi elements arise from ER membranes that are present in the bud. These two pathways of Golgi inheritance in S. cerevisiae resemble Golgi inheritance pathways in vertebrate cells

Determining protein interaction specificity of native and designed bZIP family transcription factors

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Page 428 blank. Cataloged from PDF version of thesis.Includes bibliographical references.Protein-protein interactions are important for almost all cellular functions. Knowing which proteins interact with one another is important for understanding protein function as well as for being able to disrupt their interactions. The basic leucine-zipper transcription factors (bZIPs) are a class of eukaryotic transcription factors that form either homodimers or heterodimers that bind to DNA in a site-specific manner. bZIPs are similar in sequence and structure, yet bZIP protein-protein interactions are specific, and this specificity is important for determining which DNA sites are bound. bZIP proteins have a simple structure that makes them experimentally tractable and well suited for developing models of interaction specificity. While current models perform well at being able to distinguish interactions from non-interactions, they are not fully accurate or able to predict interaction affinity. Our current understanding of protein interaction specificity is limited by the small number of large, high-quality interaction data sets that can be analyzed. For my thesis work I took a biophysical approach to experimentally measure the interactions of many native and designed bZIP and bZIP-like proteins in a high-throughput manner. The first method I used involved protein arrays containing small spots of bZIP-derived peptides immobilized on glass slides, which were probed with fluorescently labeled candidate protein partners. To improve upon this technique, I developed a solution-based FRET assay. In this experiment, two different dye-labeled versions of each protein are purified and mixed together at multiple concentrations to generate binding curves that quantify the affinity of each pair-wise interaction. Using the array assay, I identified novel interactions between human proteins and virally encoded bZIPs, characterized peptides designed to bind specifically to native bZIPs, and measured the interactions of a large set of synthetic bZIP-like coiled coils. Using the solution-based FRET assay, I quantified the bZIP interaction networks of five metazoan species and observed conservation as well as rewiring of interactions throughout evolution. Together, these studies have identified new interactions, created peptide reagents, identified sequence determinants of interaction specificity, and generated large amounts of interaction data that will help in the further understanding of bZIP protein interaction specificity.by Aaron W. Reinke.Ph.D