Richard Binari

A genome-scale shRNA resource for transgenic RNAi in Drosophila

Existing transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.

ACTIVATION OF A DROSOPHILA JANUS KINASE (JAK) CAUSES HEMATOPOIETIC NEOPLASIA AND DEVELOPMENTAL DEFECTS

In mammals, many cytokines and growth factors stimulate members of the Janus kinase (JAK) family to transduce signals for the proliferation and differentiation of various cell types, particularly in hematopoietic lineages. Mutations in the Drosophila hopscotch (hop) gene, which encodes a JAK, also cause proliferative defects. Loss‐of‐function alleles result in lethality and underproliferation of diploid tissues of the larva. A dominant gain‐of‐function allele, Tumorous‐lethal (hopTum‐l), leads to formation of melanotic tumors and hypertrophy of the larval lymph glands, the hematopoietic organs. We show that a single amino acid change in Hop is associated with the hopTum‐l mutation. Overexpression of either wild‐type hop or hopTum‐l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy indistinguishable from the original hopTum‐l mutation. In addition, overexpression of Hop in other tissues of the larva leads to pattern defects in the adult or to lethality. Finally, overexpression of either hop or hopTum‐l in Drosophila cell culture results in tyrosine phosphorylation of Hop protein. However, overexpression of hopTum‐l results in greater phosphorylation than overexpression of the wild‐type. We conclude that hopTum‐l encodes a hyperactive Hop kinase and that overactivity of Hop in lymph glands causes malignant neoplasia of Drosophila blood cells.

A Drosophila Resource of Transgenic RNAi Lines for Neurogenetics

Conditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the “VALIUM” series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.

Hierarchical rules for Argonaute loading in Drosophila

Drosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. MicroRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a byproduct of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway.

Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster

The conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site–driven RNA interference constructs have been inserted into the genome randomly using P-element–mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.

The conserved PI3′K/PTEN/Akt signaling pathway regulates both cell size and survival in Drosophila

Akt (or PKB) is an oncogene involved in the regulation of cell survival. Akt is regulated by phosphatidylinositol 3-OH kinase (PI3′K) signaling and has shown to be hyperactivated through the loss of the PTEN tumor suppressor. In Drosophila, insulin signaling as studied using the Drosophila IRS-4 homolog (Chico) has been shown to be a crucial regulator of cell size. We have studied Drosophila Akt (Dakt1) and have shown that it is also involved in the regulation of cell size. Furthermore we have performed genetic epistasis tests to demonstrate that in Drosophila, PI3′K, PTEN and Akt comprise a signaling cassette that is utilized during multiple stages of development. In addition, we show that this signaling cassette is also involved in the regulation of cell survival during embryogenesis. This study therefore establishes the evolutionary conservation of this signaling pathway in Drosophila.

A genomewide RNA interference screen for modifiers of aggregates formation by mutant huntingtin in drosophila

Protein aggregates are a common pathological feature of most neurodegenerative diseases (NDs). Understanding their formation and regulation will help clarify their controversial roles in disease pathogenesis. To date, there have been few systematic studies of aggregates formation in Drosophila, a model organism that has been applied extensively in modeling NDs and screening for toxicity modifiers. We generated transgenic fly lines that express enhanced-GFP-tagged mutant Huntingtin (Htt) fragments with different lengths of polyglutamine (polyQ) tract and showed that these Htt mutants develop protein aggregates in a polyQ-length- and age-dependent manner in Drosophila. To identify central regulators of protein aggregation, we further generated stable Drosophila cell lines expressing these Htt mutants and also established a cell-based quantitative assay that allows automated measurement of aggregates within cells. We then performed a genomewide RNA interference screen for regulators of mutant Htt aggregation and isolated 126 genes involved in diverse cellular processes. Interestingly, although our screen focused only on mutant Htt aggregation, several of the identified candidates were known previously as toxicity modifiers of NDs. Moreover, modulating the in vivo activity of hsp110 (CG6603) or tra1, two hits from the screen, affects neurodegeneration in a dose-dependent manner in a Drosophila model of Huntingtons disease. Thus, other aggregates regulators isolated in our screen may identify additional genes involved in the protein-folding pathway and neurotoxicity.

Chk2 regulates irradiation-induced, p53-mediated apoptosis in Drosophila

The tumor suppressor function of p53 has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, aberrant growth signals, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. In Drosophila melanogaster, however, the regulation modes of p53 are still unknown. Overexpression of D. melanogaster p53 (Dmp53) in the eye induced apoptosis, resulting in a small eye phenotype. This phenotype was markedly enhanced by coexpression with D. melanogaster Chk2 (DmChk2) and was almost fully rescued by coexpression with a dominant-negative (DN), kinase-dead form of DmChk2. DN DmChk2 also inhibited Dmp53-mediated apoptosis in response to DNA damage, whereas overexpression of Grapes (Grp), the Drosophila Chk1-homolog, and its DN mutant had no effect on Dmp53-induced phenotypes. DmChk2 also activated the Dmp53 transactivation activity in cultured cells. Mutagenesis of Dmp53 amino terminal Ser residues revealed that Ser-4 is critical for its responsiveness toward DmChk2. DmChk2 activates the apoptotic activity of Dmp53 and Ser-4 is required for this effect. Contrary to results in mammals, Grapes, the Drosophila Chk1-homolog, is not involved in regulating Dmp53. Chk2 may be the ancestral regulator of p53 function.

The Transgenic RNAi Project at Harvard Medical School: Resources and Validation

To facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT–qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT–qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).

Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling.

Interactome mapping and functional genomics in Drosophila reveal common and specific components of a mitogen-activated protein kinase pathway. Finding the Shared and the Specific Components Regulating MAPK Signals Even in extensively studied pathways, such as the extracellular signal–regulated kinase (ERK) pathway that is activated by receptor tyrosine kinases, there remain gaps in our knowledge. Friedman et al. combined protein-protein interaction screens with RNAi functional genomic screens in Drosophila cell lines to identify components of the ERK pathway downstream of two receptor tyrosine kinases. Their analysis suggested that these receptors may compete for some common components, in addition to using receptor-specific and cell-specific signal transduction pathways. Knockdown of several newly identified pathway regulators resulted in wing phenotypes in vivo, confirming these as components in the pathway. Detailed understanding of this pathway has clinical relevance because of its importance in both physiological and pathophysiological contexts, such as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Characterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)–Ras–extracellular signal–regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification–mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type– and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine–protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.