A Genome-Wide Map of Conserved MicroRNA Targets in C. elegans

SummaryBackgroundMetazoan miRNAs regulate protein-coding genes by binding the 3′ UTR of cognate mRNAs. Identifying targets for the 115 known C. elegans miRNAs is essential for understanding their function.ResultsBy using a new version of PicTar and sequence alignments of three nematodes, we predict that miRNAs regulate at least 10% of C. elegans genes through conserved interactions. We have developed a new experimental pipeline to assay 3′ UTR-mediated posttranscriptional gene regulation via an endogenous reporter expression system amenable to high-throughput cloning, demonstrating the utility of this system using one of the most intensely studied miRNAs, let-7. Our expression analyses uncover several new potential let-7 targets and suggest a new let-7 activity in head muscle and neurons. To explore genome-wide trends in miRNA function, we analyzed functional categories of predicted target genes, finding that one-third of C. elegans miRNAs target gene sets are enriched for specific functional annotations. We have also integrated miRNA target predictions with other functional genomic data from C. elegans.ConclusionsAt least 10% of C. elegans genes are predicted miRNA targets, and a number of nematode miRNAs seem to regulate biological processes by targeting functionally related genes. We have also developed and successfully utilized an in vivo system for testing miRNA target predictions in likely endogenous expression domains. The thousands of genome-wide miRNA target predictions for nematodes, humans, and flies are available from the PicTar website and are linked to an accessible graphical network-browsing tool allowing exploration of miRNA target predictions in the context of various functional genomic data resources

The Macronuclear Genome of \u3cem\u3eStentor coeruleus\u3c/em\u3e Reveals Tiny Introns in a Giant Cell

The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities—if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor’s cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor

Stochastic De-repression of Rhodopsins in Single Photoreceptors of the Fly Retina

The photoreceptors of the Drosophila compound eye are a classical model for studying cell fate specification. Photoreceptors (PRs) are organized in bundles of eight cells with two major types – inner PRs involved in color vision and outer PRs involved in motion detection. In wild type flies, most PRs express a single type of Rhodopsin (Rh): inner PRs express either Rh3, Rh4, Rh5 or Rh6 and outer PRs express Rh1. In outer PRs, the K50 homeodomain protein Dve is a key repressor that acts to ensure exclusive Rh expression. Loss of Dve results in de-repression of Rhodopsins in outer PRs, and leads to a wide distribution of expression levels. To quantify these effects, we introduce an automated image analysis method to measure Rhodopsin levels at the single cell level in 3D confocal stacks. Our sensitive methodology reveals cell-specific differences in Rhodopsin distributions among the outer PRs, observed over a developmental time course. We show that Rhodopsin distributions are consistent with a two-state model of gene expression, in which cells can be in either high or basal states of Rhodopsin production. Our model identifies a significant role of post-transcriptional regulation in establishing the two distinct states. The timescale for interconversion between basal and high states is shown to be on the order of days. Our results indicate that even in the absence of Dve, the Rhodopsin regulatory network can maintain highly stable states. We propose that the role of Dve in outer PRs is to buffer against rare fluctuations in this network

The nuclear transport factor CSE1 drives macronuclear volume increase and macronuclear node coalescence in Stentor coeruleus.

Stentor coeruleus provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism. When the nuclear transport factor, CSE1, is knocked down by RNAi, the shape and volume changes of the macronucleus are attenuated, and nuclear morphology is altered. CSE1 protein undergoes a dynamic relocalization correlated with nuclear shape changes, being mainly cytoplasmic prior to nuclear coalescence, and accumulating inside the macronucleus during coalescence. At the end of regeneration, CSE1 protein levels are reduced as the macronucleus returns to its pre-coalescence volume. We propose a model in which nuclear transport via CSE1 is required to increase the volume of the macronucleus, thereby decreasing the surface-to-volume ratio and driving coalescence of the nodes into a single mass

The nuclear transport factor CSE1 drives macronuclear volume increase and macronuclear node coalescence in Stentor coeruleus

Summary: Stentor coeruleus provides a unique opportunity to study how cells regulate nuclear shape because its macronucleus undergoes a rapid, dramatic, and developmentally regulated shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism. When the nuclear transport factor, CSE1, is knocked down by RNAi, the shape and volume changes of the macronucleus are attenuated, and nuclear morphology is altered. CSE1 protein undergoes a dynamic relocalization correlated with nuclear shape changes, being mainly cytoplasmic prior to nuclear coalescence, and accumulating inside the macronucleus during coalescence. At the end of regeneration, CSE1 protein levels are reduced as the macronucleus returns to its pre-coalescence volume. We propose a model in which nuclear transport via CSE1 is required to increase the volume of the macronucleus, thereby decreasing the surface-to-volume ratio and driving coalescence of the nodes into a single mass

Distribution of pairwise Rhodopsin expression.

<p>(A & B) Each point corresponds to a single PR, with the two coordinates giving the relative expression levels of two Rhodopsins. Data was pooled across all replicates at each time point. To give a sense for the density of points in different regions, each point was colored to indicate the number of points within a radius = 0.5 around it. The color bar shows the number of points indicated by each color.</p

Bimodal expression of Rh6 in R8 cells.

<p>The distribution of Rh6 levels in R8 cells shown here represents retinae at all time points. The bar graph indicates the probability density of all PRs in each expression level bin. A bimodal distribution, given by a mixture of two normal distributions, was fit to the data using maximum likelihood fitting. The means (μ) and standard deviations (σ) of the two Gaussians are shown, and the mixture proportion is such that 80% of cells express Rh6 at high levels, while 20% express Rh6 at low levels.</p

Rh3, Rh5 and Rh6 are de-repressed in <i>dve</i> mutants.

<p>(A) Three dimensional rendering of a representative confocal stack of a retina dissected at the 2 week developmental time point. Phalloidin, which stains actin, was used to visualize the rhabdomeres (green). This retina is co-stained for Rh5 (red) and Rh6 (blue). The inset shown is a zoomed-in view of the center of the retina. (B & C) Retinae were co-stained for two Rhodopsins, and representative ommatidia extracted automatically from the image stacks are shown for retinae co-stained for either Rh3–Rh6 or Rh5–Rh6 (panels B-i & C-i). Panels B-ii & C-ii show a cross section of the ommatidium in the phalloidin channel, indicating the automatically identified PR cells (R1–R6; R7/R8). Rh3 levels exhibit de-repression in outer PR cells (B-iii). Rh6 levels exhibit de-repression in outer PR cells (panels B-iv & C-iv). Rh5 levels exhibit de-repression in outer PR cells (C-iii). Scale bar is 1.5 µm (panels B) and 1.0 µm (panels C).</p