Gene silencing by double-stranded RNA.

Eukaryotes silence gene expression in the presence of double-stranded RNA homologous to the silenced gene. Silencing occurs by the targeted degradation of mRNA. Biochemical reactions that recapitulate this phenomenon generate RNA fragments of 21--23 nucleotides from the double-stranded RNA. These stably associate with an RNA endonuclease and probably serve as a discriminator to select mRNAs. Once selected, mRNAs are cleaved at sites 21--23 nucleotides apart. This mechanism, termed RNAi, has functional links to viral defense and silencing phenomena, such as cosuppression. It also functions to repress the hopping of transposable elements.

Stem cell division is regulated by the microRNA pathway.

One of the key characteristics of stem cells is their capacity to divide for long periods of time in an environment where most of the cells are quiescent. Therefore, a critical question in stem cell biology is how stem cells escape cell division stop signals. Here, we report the necessity of the microRNA (miRNA) pathway for proper control of germline stem cell (GSC) division in Drosophila melanogaster. Analysis of GSCs mutant for dicer-1 (dcr-1), the double-stranded RNaseIII essential for miRNA biogenesis, revealed a marked reduction in the rate of germline cyst production. These dcr-1 mutant GSCs exhibit normal identity but are defective in cell cycle control. On the basis of cell cycle markers and genetic interactions, we conclude that dcr-1 mutant GSCs are delayed in the G1 to S transition, which is dependent on the cyclin-dependent kinase inhibitor Dacapo, suggesting that miRNAs are required for stem cells to bypass the normal G1/S checkpoint. Hence, the miRNA pathway might be part of a mechanism that makes stem cells insensitive to environmental signals that normally stop the cell cycle at the G1/S transition.

Genome-wide survey of protein kinases required for cell cycle progression.

Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery.

The adenovirus major late transcription factor activates the rat gamma-fibrinogen promoter.

The major late transcription factor (MLTF) is a 46-kilodalton polypeptide that specifically binds to and activates transcription from the major late promoter of adenovirus. The presence of this promoter-specific transcription factor in uninfected HeLa cell extracts suggests that MLTF is also involved in the transcription of cellular genes. This report demonstrates that MLTF specifically stimulates transcription of the rat gamma-fibrinogen gene through a high-affinity binding site. Stimulation of transcription by MLTF was not dependent on the exact position of the MLTF binding site with respect either to the transcription initiation site or to adjacent promoter elements. These results suggest that one of the cellular functions of MLTF is to control gamma-fibrinogen gene expression.

Endocytosis: why not wait to deubiquitinate?

Deubiquitination by the Fat facets protein - a regulator of photoreceptor differentiation during Drosophila eye development - has been found to activate endocytosis, while ubiquitination inhibits endocytosis. Surprisingly, this is the opposite effect that ubiquitination has on endocytosis of many plasma membrane proteins.

A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor.

A simple approach has been developed for the unambiguous identification and purification of sequence-specific DNA-binding proteins solely on the basis of their ability to bind selectively to their target sequences. Four independent methods were used to identify the promoter-specific RNA polymerase II transcription factor MLTF as a 46-kilodalton (kDa) polypeptide. First, a 46-kDa protein was specifically cross-linked by UV irradiation to a body-labeled DNA fragment containing the MLTF binding site. Second, MLTF sedimented through glycerol gradients at a rate corresponding to a protein of native molecular weight 45,000 to 50,000. Third, a 46-kDa protein was specifically retained on a biotin-streptavidin matrix only when the DNA fragment coupled to the matrix contained the MLTF binding site. Finally, proteins from the most highly purified fraction which were eluted and renatured from the 44- to 48-kDa region of a sodium dodecyl sulfate-polyacrylamide gel exhibited both binding and transcription-stimulatory activities. The DNA-binding activity was purified 100,000-fold by chromatography through three conventional columns plus a DNA affinity column. Purified MLTF was characterized with respect to the kinetic and thermodynamic properties of DNA binding. These parameters indicate a high degree of occupancy of MLTF binding sites in vivo.

Heritable gene silencing in Drosophila using double-stranded RNA.

RNA-mediated interference (RNAi) is a recently discovered method to determine gene function in a number of organisms, including plants, nematodes, Drosophila, zebrafish, and mice. Injection of double-stranded RNA (dsRNA) corresponding to a single gene into organisms silences expression of the specific gene. Rapid degradation of mRNA in affected cells blocks gene expression. Despite the promise of RNAi as a tool for functional genomics, injection of dsRNA interferes with gene expression transiently and is not stably inherited. Consequently, use of RNAi to study gene function in the late stages of development has been limited. It is particularly problematic for development of disease models that reply on post-natal individuals. To circumvent this problem in Drosophila, we have developed a method to express dsRNA as an extended hairpin-loop RNA. This method has recently been successful in generating RNAi in the nematode Caenorhabditis elegans. The hairpin RNA is expressed from a transgene exhibiting dyad symmetry in a controlled temporal and spatial pattern. We report that the stably inherited transgene confers specific interference of gene expression in embryos, and tissues that give rise to adult structures such as the wings, legs, eyes, and brain. Thus, RNAi can be adapted to study late-acting gene function in Drosophila. The success of this approach in Drosophila and C. elegans suggests that a similar approach may prove useful to study gene function in higher organisms for which transgenic technology is available.

Drosophila Eye Nuclei Segmentation Based on Graph Cut and Convex Shape Prior.

The rapid advance in three-dimensional (3D) confocal imaging technologies is rapidly increasing the availability of 3D cellular images. However, the lack of robust automated methods for the extraction of cell or organelle shapes from the images is hindering researchers ability to take full advantage of the increase in experimental output. The lack of appropriate methods is particularly significant when the density of the features of interest in high, such as in the developing eye of the fruit fly. Here, we present a novel and efficient nuclei segmentation algorithm based on the combination of graph cut and convex shape prior. The main characteristic of the algorithm is that it segments nuclei foreground using a graph cut algorithm and splits overlapping or touching cell nuclei by simple convex and concavity analysis, using a convex shape assumption for nuclei contour. We evaluate the performance of our method by applying it to a library of publicly-available two-dimensional (2D) images that were hand-labeled by experts. Our algorithm yields a substantial quantitative improvement over other methods for this benchmark. For example, our method achieves a decrease of 3.2 in the Hausdorff distance and an decrease of 1.8 per slice in the merged nuclei error.

The RNAi pathway initiated by Dicer-2 in Drosophila.

Injection or expression of double-stranded RNA (dsRNA) in Drosophila serves as a trigger that causes cells to specifically cleave homologous mRNA transcripts. Our approach is to identify essential components of the RNA interference (RNAi) mechanism by isolating and characterizing mutations that cause the RNAi response to be abnormal. These studies have thus far led to the identification of seven genetic loci that encode proteins acting at various steps in the RNAi process. We have molecularly identified several of these proteins. Two are members of the Dicer family. Dicer-1 and Dicer-2 are required for short interfering RNA (siRNA)-directed mRNA cleavage by facilitating distinct steps in the assembly of the RNA-induced silencing complex (RISC). AGO2 is a RISC component that both carries out transcript cleavage and facilitates RISC maturation. Other factors appear to function as regulators of RISC assembly rather than as core factors for RNAi.

Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.

We investigated the potential of double-stranded RNA to interfere with the function of genes in Drosophila. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. In contrast, single-stranded RNA weakly interfered with gene activity. The method was used to determine the reception mechanism of the morphogen Wingless. Interference of the frizzled and Drosophila frizzled 2 genes together produced defects in embryonic patterning that mimic loss of wingless function. Interference of either gene alone had no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. Our results demonstrate that dsRNA interference can be used to analyze many aspects of gene function.

Interactions between Wingless and DFz2 during Drosophila wing development.

Drosophila Wingless (Wg) is a secreted signaling protein of the Wnt family. Mutations in the wg gene disrupt the patterning of embryonic segments and their adult derivatives. Wg protein has been shown in cell culture to functionally interact with DFz2, a receptor that is structurally related to the tissue polarity protein Frizzled (Fz). However, it has not been determined if DFz2 functions in the Wg signaling pathway during fly development. Here we demonstrate that overexpression of DFz2 increases Wg-dependent signaling to induce ectopic margin bristle formation in developing Drosophila wings. Overexpression of a truncated form of DFz2 acts in a dominant-negative manner to block Wg signaling at the wing margin, and this block is rescued by co-expression of full-length DFz2 but not full-length Fz. Our results suggest that DFz2 and not Fz acts in the Wg signaling pathway for wing margin development. However, a truncated form of Fz also blocks Wg signaling in embryo and wing margin development, and the truncated form of DFz2 affects ommatidial polarity during eye development. These observations suggest that a single dominant-negative form of Fz or DFz2 can block more than one type of Wnt signaling pathway and imply that truncated proteins of the Fz family lose some aspect of signaling specificity.

seven in absentia, a gene required for specification of R7 cell fate in the Drosophila eye.

Signals from directly adjacent cells are critical for the development of the R7 photoreceptor cell in the Drosophila eye. We have identified a gene, seven in absentia (sina), for which loss of function results in the R7 precursor cell adopting the fate of a nonneuronal cell type. Function of the sina gene is required only in R7 for correct R7 cell development. The sina transcription unit is located within the intron of a gene that encodes an R7 cell-specific opsin. The sina protein, which has a potential metal binding domain, is localized in the nuclei of several ommatidial precursor cells including R7, and sina expression in R7 appears before R7 overtly begins to differentiate. These data indicate that the sina gene product is necessary at a stage in the determination of R7 cell fate when R7 receives and interprets developmental signals from neighboring cells, and possibly acts by regulating gene expression.

The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination.

Roughened is a dominant mutation of D. melanogaster that disrupts eye development. The majority of the ommatidia in the adult eye lack a single photoreceptor cell, which is most commonly the R7 cell. The Roughened mutation disrupts the early stages of photoreceptor cell determination. Roughened is a dominant gain-of-function mutation that results from a single amino acid change (Phe157 to Leu) in the Drosophila Rap1 protein. Loss of function Rap1 mutations are lethal. Drosophila Rap1 protein is 88% identical to human rap1A/K-rev1 protein, a putative antagonist of ras action.

Evolution of gene position: chromosomal arrangement and sequence comparison of the Drosophila melanogaster and Drosophila virilis sina and Rh4 genes.

The seven in absentia (sina) gene of Drosophila encodes a nuclear protein required for normal eye development. In Drosophila melanogaster, the sina gene is located within an intron of the Rh4 opsin gene. We examine here the nucleotide sequences and chromosomal arrangements of these genes in Drosophila virilis. An interspecies comparison between D. melanogaster and D. virilis reveals that the protein-coding sequences of the sina and Rh4 genes are highly conserved, but the relative chromosomal position and structural arrangement of these genes differ between the two species. In particular, the sina and Rh4 genes are widely separated in D. virilis, and there is no intron in the Rh4 gene. Our results suggest that the Rh4 gene was translocated to another chromosomal location by a retrotransposition event.

Identification of genes that interact with the sina gene in Drosophila eye development.

The sina gene encodes a nuclear protein that is required for the correct development of R7 photoreceptor cells in the Drosophila eye. We conducted a genetic screen for mutations that reduce the activity of sina and found mutations that define nine genes whose products may be required for normal sina activity. Three of these genes also appear to be essential for signaling by the Sevenless-Ras pathway in R7 cells, of which one gene corresponds to the rolled locus (rl). The rl gene is known to encode a mitogen-activated protein kinase necessary for signaling by Ras. These results suggest that the products of these three genes may participate in a signaling pathway involving both Ras and Sina, possibly by functionally linking these two proteins.

frizzled regulates mirror-symmetric pattern formation in the Drosophila eye.

Coordinated morphogenesis of ommatidia during Drosophila eye development establishes a mirror-image symmetric pattern across the entire eye bisected by an anteroposterior equator. We have investigated the mechanisms by which this pattern formation occurs and our results suggest that morphogenesis is coordinated by a graded signal transmitted bidirectionally from the presumptive equator to the dorsal and ventral poles. This signal is mediated by frizzled, which encodes a cell surface transmembrane protein. Mosaic analysis indicates that frizzled acts non-autonomously in an equatorial to polar direction. It also indicates that relative levels of frizzled in photoreceptor cells R3 and R4 of each ommatidium affect their positional fate choices such that the cell with greater frizzled activity becomes an R3 cell and the cell with less frizzled activity becomes an R4 cell. Moreover, this bias affects the choice an ommatidium makes as to which direction to rotate. Equator-outwards progression of elav expression and expression of the nemo gene in the morphogenetic furrow are regulated by frizzled, which itself is dynamically expressed about the morphogenetic furrow. We propose that frizzled mediates a bidirectional signal emanating from the equator.

Isolation of three proteins that bind to mammalian RNA polymerase II.

We have used affinity chromatography on columns containing immobilized calf thymus RNA polymerase II to isolate three phosphoproteins (RAP72, RAP38, and RAP30) that bind directly to RNA polymerase II. All could be isolated from cell nuclei, and all three could be detected in mouse and human tissue culture cell lines, but only RAP38 and RAP30 have so far been isolated from calf thymus. RAP38 stimulates nonspecific transcription of native DNA templates by RNA polymerase II in the presence of Mn2+; it appears to be similar or identical to SII, a previously identified RNA polymerase II stimulatory factor (Nakanishi, Y., Mitsuhashi, Y., Sekimizu, K., Yokoi, H., Tanaka, Y., Horikoshi, M., and Natori, S. (1981) FEBS Lett. 130, 69-72). Unlike RAP38, RAP72 and RAP30 do not affect nonspecific transcription by RNA polymerase II. However, RAP30 may have a role in regulating some alterations of transcription that accompany cellular differentiation; RAP30 is partially dephosphorylated when murine erythroleukemia cells are induced with dimethyl sulfoxide to undergo terminal erythroid differentiation. We suggest that phosphate groups in RNA polymerase II-binding proteins may regulate transcription by modulating the interaction of RNA polymerase II with other regulatory proteins that possess sequence recognition specificity.

An RNA polymerase II transcription factor binds to an upstream element in the adenovirus major late promoter.

A gel electrophoresis DNA binding assay has been used to identify proteins in HeLa cell extracts that specifically bind to the major late promoter of adenovirus. A major late promoter transcription factor MLTF has been detected as a discrete protein-DNA complex. MLTF binds specifically and with high affinity to sequences upstream of the TATA box of the major late promoter. This factor protects a 17 bp (-50 to -66) region in a DNAase I footprinting assay. The same region has been shown to be important for efficient transcription from the major late promoter both in vivo and in vitro. MLTF stimulates in vitro transcription only from a template containing this upstream region. The binding, footprinting, and transcription-stimulatory activities of MLTF cofractionate through two chromatographic steps. These results suggest that direct binding of MLTF to an upstream element activates transcription from the major late promoter.

The major late transcription factor binds to and activates the mouse metallothionein I promoter.

Human (HeLa) cells contain a protein, MLTF, which specifically binds to a DNA sequence in the adenovirus 2 major late promoter and activates transcription of that promoter. The presence of MLTF in uninfected cells suggests that this factor contributes to the transcription of some cellular genes. We find that MLTF binds in a sequence-specific manner to the 5'-flanking region of the mouse metallothionein I (mMTI) gene. Binding was localized between -101 and -94 (relative to the initiation site at +1) by DNA-binding gel electrophoresis assay and DNA methylation interference analysis. As in adenovirus, binding occurred in a region containing the sequence CPuCGTGAC. Deletion of this sequence both eliminated the binding of MLTF and produced a fourfold reduction in transcriptional efficiency in vitro. In contrast to the intact promoter, transcription from the deletion mutant promoter was not stimulated by addition of purified MLTF to an in vitro reconstituted reaction. These results suggest that MLTF contributes to the transcription of cellular genes.

Formation of transcription preinitiation complexes with an amanitin-resistant RNA polymerase II.

Accurate transcription by RNA polymerase II has been shown to require multiple factors which participate in a number of intermediate steps prior to initiation. The last detectable step before initiation is the formation of an activated or rapid start complex which is template-associated. In this study we used two phenotypically distinguishable forms of mammalian RNA polymerase II to examine the requirement for specific factors in the formation of the activated complex. RNA polymerase II was purified from a mutant cell line which was resistant to levels of alpha-amanitin that are toxic for normal cells. When added to a polymerase-dependent transcription system consisting of HeLa factors and adenovirus DNA, the mutant polymerase accurately transcribed the template in an amanitin-resistant fashion. This amanitin-resistant transcription was competitively inhibited when wild-type polymerase was also added to the system. Preincubation of amanitin-resistant polymerase with DNA and factors produced a polymerase-associated complex, defined by its resistance to exchange for the amanitin-sensitive polymerase. Complex formation required the presence of polymerase, DNA, and the HeLa factors during the preincubation but did not require the presence of nucleotides. Complexes were template-associated, as shown by their inability to exchange onto a second template. Thus, prior to initiation, RNA polymerase II forms a stable association with the DNA template in an activated complex.