Genome-scale RNAi profiling of cell division in human tissue culture cells.

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.

Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis.

Endocytosis is a key cellular process, encompassing different entry routes and endocytic compartments. To what extent endocytosis is subjected to high-order regulation by the cellular signalling machinery remains unclear. Using high-throughput RNA interference and automated image analysis, we explored the function of human kinases in two principal types of endocytosis: clathrin- and caveolae/raft-mediated endocytosis. We monitored this through infection of vesicular stomatitis virus, simian virus 40 and transferrin trafficking, and also through cell proliferation and apoptosis assays. Here we show that a high number of kinases are involved in endocytosis, and that each endocytic route is regulated by a specific kinase subset. Notably, one group of kinases exerted opposite effects on the two endocytic routes, suggesting coordinate regulation. Our analysis demonstrates that signalling functions such as those controlling cell adhesion, growth and proliferation, are built into the machinery of endocytosis to a much higher degree than previously recognized.

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

Genome-wide resources of endoribonuclease-prepared short interfering RNAs for specific loss-of-function studies.

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.

A genome-wide visual screen reveals a role for sphingolipids and ergosterol in cell surface delivery in yeast.

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.