Cell division is fundamental for all organisms. Here we report a genome-scale RNA-mediated interference screen in HeLa cells designed to identify human genes that are important for cell division. We have used a library of endoribonuclease-prepared short interfering RNAs for gene silencing and have used DNA content analysis to identify genes that induced cell cycle arrest or altered ploidy on silencing. Validation and secondary assays were performed to generate a nine-parameter loss-of-function phenoprint for each of the genes. These phenotypic signatures allowed the assignment of genes to specific functional classes by combining hierarchical clustering, cross-species analysis and proteomic data mining. We highlight the richness of our dataset by ascribing novel functions to genes in mitosis and cytokinesis. In particular, we identify two evolutionarily conserved transcriptional regulatory networks that govern cytokinesis. Our work provides an experimental framework from which the systematic analysis of novel genes necessary for cell division in human cells can begin.

A systematic search for human ribosome biogenesis factors shows conservation of many aspects of eukaryotic ribosome synthesis with the well-studied process in yeast and identifies an export route of 60S subunits that is specific for higher eukaryotes.

RNA interference (RNAi) has become an important technique for loss-of-gene-function studies in mammalian cells. To achieve reliable results in an RNAi experiment, efficient and specific silencing triggers are required. Here we present genome-wide data sets for the production of endoribonuclease-prepared short interfering RNAs (esiRNAs) for human, mouse and rat. We used an algorithm to predict the optimal region for esiRNA synthesis for every protein-coding gene of these three species. We created a database, RiDDLE, for retrieval of target sequences and primer information. To test this in silico resource experimentally, we generated 16,242 esiRNAs that can be used for RNAi screening in human cells. Comparative analyses with chemically synthesized siRNAs demonstrated a high silencing efficacy of esiRNAs and a 12-fold reduction of downregulated off-target transcripts as detected by microarray analysis. Hence, the presented esiRNA libraries offer an efficient, cost-effective and specific alternative to presently available mammalian RNAi resources.

Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrp1p), known membrane traffic regulators (Kes1p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting.exocytosis ͉ lipid rafts ͉ Saccharomyces cerevisiae ͉ sorting ͉ Golgi T he mechanisms responsible for sorting proteins to the cell surface from the Golgi complex are poorly understood in eukaryotic cells. The trans-Golgi network (TGN) has been recognized as a major hub for sorting (1). However, there is also evidence that sorting occurs in endosomes (2). In polarized cells such as epithelial cells and neurons, biosynthetic cargo is delivered to separate membrane domains by pathways employing different sorting principles (3,4). Recent work has demonstrated that yeast cells also have at least two separate routes to the cell surface (5-8). Little is known about the genes that are responsible for sorting and packaging surface cargo into different transport containers. Previous screens aimed at identifying this machinery relied, for example, on major growth defects and the internal accumulation of invertase, which has been later shown to be transported by the minor pathway to the plasma membrane (7,8). These screens have mainly identified mutants that blocked endoplasmic reticulum (ER)-to-Golgi transport and delivery to the plasma membrane (9, 10). However, mutations in regulators of post-Golgi sorting and vesicle formation with few exceptions have not been detected by such screens, probably because a block in one transport route to the cell surface can be rescued by partial rerouting from the affected to the undisturbed pathway (7,8).Here we describe a visual screening procedure devised to circumvent this problem. We aimed at developing an assay sensitive enough to detect sorting defects within the secretory pathway and applicable to genome-wide screening. The screen takes advantage of the systematic yeast knockout array (11), which should contain the nonessential genes responsible for regulating cargo entry into specialized, partially redundant pathways. The results of this genome-wide screen demonstrate the suitability of our visual screening approach for identifying regulators of sorting and vesicle formation involved in surface delivery of biosynthetic cargo. Table 1. Images (GFP and DIC) ...

Here we show the crucial role of MPP1 in lateral membrane ordering/organization in HEL cells (derived from erythroid precursors). Biochemical analyses showed that inhibition of MPP1 palmitoylation or silencing of the MPP1 gene led to a dramatic decrease in the DRM fraction. This was accompanied by a reduction of membrane order as shown by fluorescence-lifetime imaging microscopy (FLIM) analyses. Furthermore, MPP1 knockdown significantly affects the activation of MAP-kinase signaling via raft-dependent RTK (receptor tyrosine kinase) receptors, indicating the importance of MPP1 for lateral membrane organization. In conclusion, palmitoylation of MPP1 appears to be at least one of the mechanisms controlling lateral organization of the erythroid cell membrane. Thus, this study, together with our recent results on erythrocytes, reported elsewhere (Łach et al., J. Biol. Chem., 2012, 287, 18974-18984), points to a new role for MPP1 and presents a novel linkage between membrane raft organization and protein palmitoylation.