Kristin C. Gunsalus

Combinatorial microRNA target predictions

MicroRNAs are small noncoding RNAs that recognize and bind to partially complementary sites in the 3′ untranslated regions of target genes in animals and, by unknown mechanisms, regulate protein production of the target transcript. Different combinations of microRNAs are expressed in different cell types and may coordinately regulate cell-specific target genes. Here, we present PicTar, a computational method for identifying common targets of microRNAs. Statistical tests using genome-wide alignments of eight vertebrate genomes, PicTars ability to specifically recover published microRNA targets, and experimental validation of seven predicted targets suggest that PicTar has an excellent success rate in predicting targets for single microRNAs and for combinations of microRNAs. We find that vertebrate microRNAs target, on average, roughly 200 transcripts each. Furthermore, our results suggest widespread coordinate control executed by microRNAs. In particular, we experimentally validate common regulation of Mtpn by miR-375, miR-124 and let-7b and thus provide evidence for coordinate microRNA control in mammals.

Full-genome RNAi profiling of early embryogenesis in Caenorhabditis elegans

A key challenge of functional genomics today is to generate well-annotated data sets that can be interpreted across different platforms and technologies. Large-scale functional genomics data often fail to connect to standard experimental approaches of gene characterization in individual laboratories. Furthermore, a lack of universal annotation standards for phenotypic data sets makes it difficult to compare different screening approaches. Here we address this problem in a screen designed to identify all genes required for the first two rounds of cell division in the Caenorhabditis elegans embryo. We used RNA-mediated interference to target 98% of all genes predicted in the C. elegans genome in combination with differential interference contrast time-lapse microscopy. Through systematic annotation of the resulting movies, we developed a phenotypic profiling system, which shows high correlation with cellular processes and biochemical pathways, thus enabling us to predict new functions for previously uncharacterized genes.

Unlocking the secrets of the genome

Despite the successes of genomics, little is known about how genetic information produces complex organisms. A look at the crucial functional elements of fly and worm genomes could change that. The National Human Genome Research Institutes modENCODE project (the model organism ENCyclopedia Of DNA Elements) was set up in 2007 with the goal of identifying all the sequence-based functional elements in the genomes of two important experimental organisms, Caenorhabditis elegans and Drosophila melanogaster. Armed with modENCODE data, geneticists will be able to undertake the comprehensive molecular studies of regulatory networks that hold the key to how complex multicellular organisms arise from the list of instructions coded in the genome. In this issue, modENCODE team members outline their plan of campaign. Data from the project are to be made available on http://www.modencode.org and elsewhere as the work progresses.

microRNA Target Predictions across Seven Drosophila Species and Comparison to Mammalian Targets

microRNAs are small noncoding genes that regulate the protein production of genes by binding to partially complementary sites in the mRNAs of targeted genes. Here, using our algorithm PicTar, we exploit cross-species comparisons to predict, on average, 54 targeted genes per microRNA above noise in Drosophila melanogaster. Analysis of the functional annotation of target genes furthermore suggests specific biological functions for many microRNAs. We also predict combinatorial targets for clustered microRNAs and find that some clustered microRNAs are likely to coordinately regulate target genes. Furthermore, we compare microRNA regulation between insects and vertebrates. We find that the widespread extent of gene regulation by microRNAs is comparable between flies and mammals but that certain microRNAs may function in clade-specific modes of gene regulation. One of these microRNAs (miR-210) is predicted to contribute to the regulation of fly oogenesis. We also list specific regulatory relationships that appear to be conserved between flies and mammals. Our findings provide the most extensive microRNA target predictions in Drosophila to date, suggest specific functional roles for most microRNAs, indicate the existence of coordinate gene regulation executed by clustered microRNAs, and shed light on the evolution of microRNA function across large evolutionary distances. All predictions are freely accessible at our searchable Web site http://pictar.bio.nyu.edu.

Predictive models of molecular machines involved in Caenorhabditis elegans early embryogenesis

Although numerous fundamental aspects of development have been uncovered through the study of individual genes and proteins, system-level models are still missing for most developmental processes. The first two cell divisions of Caenorhabditis elegans embryogenesis constitute an ideal test bed for a system-level approach. Early embryogenesis, including processes such as cell division and establishment of cellular polarity, is readily amenable to large-scale functional analysis. A first step toward a system-level understanding is to provide ‘first-draft’ models both of the molecular assemblies involved and of the functional connections between them. Here we show that such models can be derived from an integrated gene/protein network generated from three different types of functional relationship: protein interaction, expression profiling similarity and phenotypic profiling similarity, as estimated from detailed early embryonic RNA interference phenotypes systematically recorded for hundreds of early embryogenesis genes. The topology of the integrated network suggests that C. elegans early embryogenesis is achieved through coordination of a limited set of molecular machines. We assessed the overall predictive value of such molecular machine models by dynamic localization of ten previously uncharacterized proteins within the living embryo.

Protein NMR spectroscopy in structural genomics.

Protein NMR spectroscopy provides an important complement to X-ray crystallography for structural genomics, both for determining three-dimensional protein structures and in characterizing their biochemical and biophysical functions.

A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation.

A Drosophila melanogaster homologue of Caenorhabditis elegans par-1 acts at an early step in embryonic-axis formation

A High-Resolution C. elegans Essential Gene Network Based on Phenotypic Profiling of a Complex Tissue

High-content screening for gene profiling has generally been limited to single cells. Here, we explore an alternative approach-profiling gene function by analyzing effects of gene knockdowns on the architecture of a complex tissue in a multicellular organism. We profile 554 essential C. elegans genes by imaging gonad architecture and scoring 94 phenotypic features. To generate a reference for evaluating methods for network construction, genes were manually partitioned into 102 phenotypic classes, predicting functions for uncharacterized genes across diverse cellular processes. Using this classification as a benchmark, we developed a robust computational method for constructing gene networks from high-content profiles based on a network context-dependent measure that ranks the significance of links between genes. Our analysis reveals that multi-parametric profiling in a complex tissue yields functional maps with a resolution similar to genetic interaction-based profiling in unicellular eukaryotes-pinpointing subunits of macromolecular complexes and components functioning in common cellular processes.

Robotic Cloning and Protein Production Platform of the Northeast Structural Genomics Consortium

In this chapter we describe the core Protein Production Platform of the Northeast Structural Genomics Consortium (NESG) and outline the strategies used for producing high-quality protein samples using Escherichia coli host vectors. The platform is centered on 6X-His affinity-tagged protein constructs, allowing for a similar purification procedure for most targets, and the implementation of high-throughput parallel methods. In most cases, these affinity-purified proteins are sufficiently homogeneous that a single subsequent gel filtration chromatography step is adequate to produce protein preparations that are greater than 98% pure. Using this platform, over 1000 different proteins have been cloned, expressed, and purified in tens of milligram quantities over the last 36-month period (see Summary Statistics for All Targets, ). Our experience using a hierarchical multiplex expression and purification strategy, also described in this chapter, has allowed us to achieve success in producing not only protein samples but also many three-dimensional structures. As of December 2004, the NESG Consortium has deposited over 145 new protein structures to the Protein Data Bank (PDB); about two-thirds of these protein samples were produced by the NESG Protein Production Facility described here. The methods described here have proven effective in producing quality samples of both eukaryotic and prokaryotic proteins. These improved robotic and?or parallel cloning, expression, protein production, and biophysical screening technologies will be of broad value to the structural biology, functional proteomics, and structural genomics communities.

Feo, the Drosophila Homolog of PRC1, Is Required for Central-Spindle Formation and Cytokinesis

We performed a functional analysis of fascetto (feo), a Drosophila gene that encodes a protein homologous to the Ase1p/PRC1/MAP65 conserved family of microtubule-associated proteins (MAPs). These MAPs are enriched at the spindle midzone in yeast and mammals and at the fragmoplast in plants, and are essential for the organization and function of these microtubule arrays. Here we show that the Feo protein is specifically enriched at the central-spindle midzone and that its depletion either by mutation or by RNAi results in aberrant central spindles. In Feo-depleted cells, late anaphases showed normal overlap of the antiparallel MTs at the cell equator, but telophases displayed thin MT bundles of uniform width instead of robust hourglass-shaped central spindles. These thin central spindles exhibited diffuse localizations of both the Pav and Asp proteins, suggesting that these spindles comprise improperly oriented MTs. Feo-depleted cells also displayed defects in the contractile apparatus that correlated with those in the central spindle; late anaphase cells formed regular contractile structures, but these structures did not constrict during telophase, leading to failures in cytokinesis. The phenotype of Feo-depleted telophases suggests that Feo interacts with the plus ends of central spindle MTs so as to maintain their precise interdigitation during anaphase-telophase MT elongation and antiparallel sliding.