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Background

Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated.

Results

Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols.

Conclusions

Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens.

Targeted gene silencing by RNA interference allows the study of gene function in plants and animals. In cell culture and small animal models, genetic screens can be performed—even tissue-specifically in Drosophila—with genome-wide RNAi libraries. However, a major problem with the use of RNAi approaches is the unavoidable false-positive error caused by off-target effects. Until now, this is minimized by computational RNAi design, comparing RNAi to the mutant phenotype if known, and rescue with a presumed ortholog. The ultimate proof of specificity would be to restore expression of the same gene product in vivo. Here, we present a simple and efficient method to rescue the RNAi-mediated knockdown of two independent genes in Drosophila. By exploiting the degenerate genetic code, we generated Drosophila RNAi Escape Strategy Construct (RESC) rescue proteins containing frequent silent mismatches in the complete RNAi target sequence. RESC products were no longer efficiently silenced by RNAi in cell culture and in vivo. As a proof of principle, we rescue the RNAi-induced loss of function phenotype of the eye color gene white and tracheal defects caused by the knockdown of the heparan sulfate proteoglycan syndecan. Our data suggest that RESC is widely applicable to rescue and validate ubiquitous or tissue-specific RNAi and to perform protein structure–function analysis.

A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.

Gene silencing using RNA interference (RNAi) has become a prominent biological tool for gene annotation, pathway analysis, and target discovery in mammalian cells. High-throughput screens conducted using whole-genome siRNA libraries have uncovered rich sets of new genes involved in a variety of biological processes and cellular models of disease. However, high-throughput RNAi screening is not yet a mainstream tool in life science research because current screening platforms are expensive and onerous. Miniaturizing the RNAi screening platform to reduce cost and increase throughput will enable its widespread use and harness its potential for rapid genome annotation. With this aim, we have combined semi-conductor microfabrication and nanolitre dispensing techniques to develop miniaturized electroporation-ready microwell arrays loaded with siRNA molecules in which multiplexed gene knockdown can be achieved. Arrays of microwells are created using high-aspect ratio biocompatible photoresists on optically transparent and conductive Indium-Tin Oxide (ITO) substrates with integrated micro-electrodes to enable in situ electroporation. Non-contact inkjet microarraying allows precise dispensing of nanolitre volumes into the microwell structures. We have achieved parallel electroporation of multiple mammalian cells cultured in these microwell arrays and observed efficient knockdown of genes with surface-bound, printed siRNAs. Further integration of microfabrication and non-contact nanolitre dispensing techniques described here may enable single-substrate whole-genome siRNA screening in mammalian cells.

Background

RNA interference (RNAi) becomes an increasingly important and effective genetic tool to study the function of target genes by suppressing specific genes of interest. This system approach helps identify signaling pathways and cellular phase types by tracking intensity and/or morphological changes of cells. The traditional RNAi screening scheme, in which one siRNA is designed to knockdown one specific mRNA target, needs a large library of siRNAs and turns out to be time-consuming and expensive.

Results

In this paper, we propose a conceptual model, called compressed sensing RNAi (csRNAi), which employs a unique combination of group of small interfering RNAs (siRNAs) to knockdown a much larger size of genes. This strategy is based on the fact that one gene can be partially bound with several small interfering RNAs (siRNAs) and conversely, one siRNA can bind to a few genes with distinct binding affinity. This model constructs a multi-to-multi correspondence between siRNAs and their targets, with siRNAs much fewer than mRNA targets, compared with the conventional scheme. Mathematically this problem involves an underdetermined system of equations (linear or nonlinear), which is ill-posed in general. However, the recently developed compressed sensing (CS) theory can solve this problem. We present a mathematical model to describe the csRNAi system based on both CS theory and biological concerns. To build this model, we first search nucleotide motifs in a target gene set. Then we propose a machine learning based method to find the effective siRNAs with novel features, such as image features and speech features to describe an siRNA sequence. Numerical simulations show that we can reduce the siRNA library to one third of that in the conventional scheme. In addition, the features to describe siRNAs outperform the existing ones substantially.

Conclusions

This csRNAi system is very promising in saving both time and cost for large-scale RNAi screening experiments which may benefit the biological research with respect to cellular processes and pathways.

RNA interference (RNAi) is an intracellular mechanism for post-transcriptional gene silencing that is frequently used to study gene function. RNAi is initiated by short interfering RNA (siRNA) of ∼21 nt in length, either generated from the double-stranded RNA (dsRNA) by using the enzyme Dicer or introduced experimentally. Following association with an RNAi silencing complex, siRNA targets mRNA transcripts that have sequence identity for destruction. A phenotype resulting from this knockdown of expression may inform about the function of the targeted gene. However, ‘off-target effects’ compromise the specificity of RNAi if sequence identity between siRNA and random mRNA transcripts causes RNAi to knockdown expression of non-targeted genes. The complete off-target effects must be investigated systematically on each gene in a genome by adjusting a group of parameters, which is too expensive to conduct experimentally and motivates a study in silico. This computational study examined the potential for off-target effects of RNAi, employing the genome and transcriptome sequence data of Homo sapiens, Caenorhabditis elegans and Schizosaccharomyces pombe. The chance for RNAi off-target effects proved considerable, ranging from 5 to 80% for each of the organisms, when using as parameter the exact identity between any possible siRNA sequences (arbitrary length ranging from 17 to 28 nt) derived from a dsRNA (range 100–400 nt) representing the coding sequences of target genes and all other siRNAs within the genome. Remarkably, high-sequence specificity and low probability for off-target reactivity were optimally balanced for siRNA of 21 nt, the length observed mostly in vivo. The chance for off-target RNAi increased (although not always significantly) with greater length of the initial dsRNA sequence, inclusion into the analysis of available untranslated region sequences and allowing for mismatches between siRNA and target sequences. siRNA sequences from within 100 nt of the 5′ termini of coding sequences had low chances for off-target reactivity. This may be owing to coding constraints for signal peptide-encoding regions of genes relative to regions that encode for mature proteins. Off-target distribution varied along the chromosomes of C.elegans, apparently owing to the use of more unique sequences in gene-dense regions. Finally, biological and thermodynamical descriptors of effective siRNA reduced the number of potential siRNAs compared with those identified by sequence identity alone, but off-target RNAi remained likely, with an off-target error rate of ∼10%. These results also suggest a direction for future in vivo studies that could both help in calibrating true off-target rates in living organisms and also in contributing evidence toward the debate of whether siRNA efficacy is correlated with, or independent of, the target molecule. In summary, off-target effects present a real but not prohibitive concern that should be considered for RNAi experiments.

RNA interference (RNAi) has emerged as a powerful tool to generate loss-of-function phenotypes in a variety of organisms. Combined with the sequence information of almost completely annotated genomes, RNAi technologies have opened new avenues to conduct systematic genetic screens for every annotated gene in the genome. As increasing large datasets of RNAi-induced phenotypes become available, an important challenge remains the systematic integration and annotation of functional information. Genome-wide RNAi screens have been performed both in Caenorhabditis elegans and Drosophila for a variety of phenotypes and several RNAi libraries have become available to assess phenotypes for almost every gene in the genome. These screens were performed using different types of assays from visible phenotypes to focused transcriptional readouts and provide a rich data source for functional annotation across different species. The GenomeRNAi database provides access to published RNAi phenotypes obtained from cell-based screens and maps them to their genomic locus, including possible non-specific regions. The database also gives access to sequence information of RNAi probes used in various screens. It can be searched by phenotype, by gene, by RNAi probe or by sequence and is accessible at

Background

In many eukaryotic cells, double-stranded RNA (dsRNA) triggers RNA interference (RNAi), the specific degradation of RNA of homologous sequence. RNAi is now a major tool for reverse-genetics projects, including large-scale high-throughput screens. Recent reports have questioned the specificity of RNAi, raising problems in interpretation of RNAi-based experiments.

Results

Using the protozoan Trypanosoma brucei as a model, we designed a functional complementation assay to ascertain that phenotypic effect(s) observed upon RNAi were due to specific silencing of the targeted gene. This was applied to a cytoskeletal gene encoding the paraflagellar rod protein 2 (TbPFR2), whose product is essential for flagellar motility. We demonstrate the complementation of TbPFR2, silenced via dsRNA targeting its UTRs, through the expression of a tagged RNAi-resistant TbPFR2 encoding a protein that could be immunolocalized in the flagellum. Next, we performed a functional complementation of TbPFR2, silenced via dsRNA targeting its coding sequence, through heterologous expression of the TbPFR2 orthologue gene from Trypanosoma cruzi: the flagellum regained its motility.

Conclusions

This work shows that functional complementation experiments can be readily performed in order to ascertain that phenotypic effects observed upon RNAi experiments are indeed due to the specific silencing of the targetted gene. Further, the results described here are of particular interest when reverse genetics studies cannot be easily achieved in organisms not amenable to RNAi. In addition, our strategy should constitute a firm basis to elaborate functional-dissection studies of genes from other organisms.

Background

The recently developed RNA interference (RNAi) technology has created an unprecedented opportunity which allows the function of individual genes in whole organisms or cell lines to be interrogated at genome-wide scale. However, multiple issues, such as off-target effects or low efficacies in knocking down certain genes, have produced RNAi screening results that are often noisy and that potentially yield both high rates of false positives and false negatives. Therefore, integrating RNAi screening results with other information, such as protein-protein interaction (PPI), may help to address these issues.

Results

By analyzing 24 genome-wide RNAi screens interrogating various biological processes in Drosophila, we found that RNAi positive hits were significantly more connected to each other when analyzed within a protein-protein interaction network, as opposed to random cases, for nearly all screens. Based on this finding, we developed a network-based approach to identify false positives (FPs) and false negatives (FNs) in these screening results. This approach relied on a scoring function, which we termed NePhe, to integrate information obtained from both PPI network and RNAi screening results. Using a novel rank-based test, we compared the performance of different NePhe scoring functions and found that diffusion kernel-based methods generally outperformed others, such as direct neighbor-based methods. Using two genome-wide RNAi screens as examples, we validated our approach extensively from multiple aspects. We prioritized hits in the original screens that were more likely to be reproduced by the validation screen and recovered potential FNs whose involvements in the biological process were suggested by previous knowledge and mutant phenotypes. Finally, we demonstrated that the NePhe scoring system helped to biologically interpret RNAi results at the module level.

Conclusion

By comprehensively analyzing multiple genome-wide RNAi screens, we conclude that network information can be effectively integrated with RNAi results to produce suggestive FPs and FNs, and to bring biological insight to the screening results.

SUMMARY

A key event in Wnt signaling is conversion of TCF/Lef from a transcriptional repressor to an activator, yet how this switch occurs is not well understood. Here, we report an unanticipated role for X-linked Inhibitor of Apoptosis (XIAP) in regulating this critical Wnt signaling event that is independent of its anti-apoptotic function. We identified DIAP1 as a positive regulator of Wingless signaling in a Drosophila S2 cell-based RNAi screen. XIAP, its vertebrate homolog, is similarly required for Wnt signaling in cultured mammalian cells and in Xenopus embryos, indicating evolutionary conservation of function. Upon Wnt pathway activation, XIAP is recruited to TCF/Lef where it mono-ubiquitylates Groucho(Gro)/TLE. This modification decreases affinity of Gro/TLE for TCF/Lef. Our data reveal a transcriptional switch involving XIAP-mediated ubiquitylation of Gro/TLE that facilitates its removal from TCF/Lef, thus allowing β-catenin-TCF/Lef complex assembly and initiation of a Wnt-specific transcriptional program.

Motivation: Off-target activity commonly exists in RNA interference (RNAi) screens and often generates false positives. Existing analytic methods for addressing the off-target effects are demonstrably inadequate in RNAi confirmatory screens.

Results: Here, we present an analytic method assessing the collective activity of multiple short interfering RNAs (siRNAs) targeting a gene. Using this method, we can not only reduce the impact of off-target activities, but also evaluate the specific effect of an siRNA, thus providing information about potential off-target effects. Using in-house RNAi screens, we demonstrate that our method obtains more reasonable and sensible results than current methods such as the redundant siRNA activity (RSA) method, the RNAi gene enrichment ranking (RIGER) method, the frequency approach and the t-test.

Contact: xiaohua_zhang@merck.com

Supplementary information: Supplementary data are available at Bioinformatics online.

RNA interference (RNAi) leads to sequence-specific knockdown of gene function. The approach can be used in large-scale screens to interrogate function in various model organisms and an increasing number of other species. Genome-scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C. elegans. High-throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data, including high-content image data. The results of large-scale RNAi screens have already proved useful, leading to new understandings of gene function relevant to topics such as infection, cancer, obesity and aging. Nevertheless, important caveats apply and should be taken into consideration when developing or interpreting RNAi screens. Some level of false discovery is inherent to high-throughput approaches and specific to RNAi screens, false discovery due to off-target effects (OTEs) of RNAi reagents remains a problem. The need to improve our ability to use RNAi to elucidate gene function at large scale and in additional systems continues to be addressed through improved RNAi library design, development of innovative computational and analysis tools and other approaches.

RNA interference is a powerful method to understand gene function, especially when conducted at a whole-genome scale and in a quantitative context. In C. elegans, gene function can be knocked down simply and efficiently by feeding worms with bacteria expressing a dsRNA corresponding to a specific gene 1. While the creation of libraries of RNAi clones covering most of the C. elegans genome 2,3 opened the way for true functional genomic studies (see for example 4-7), most established methods are laborious. Moy and colleagues have developed semi-automated protocols that facilitate genome-wide screens 8. The approach relies on microscopic imaging and image analysis.

Here we describe an alternative protocol for a high-throughput genome-wide screen, based on robotic handling of bacterial RNAi clones, quantitative analysis using the COPAS Biosort (Union Biometrica (UBI)), and an integrated software: the MBioLIMS (Laboratory Information Management System from Modul-Bio) a technology that provides increased throughput for data management and sample tracking. The method allows screens to be conducted on solid medium plates. This is particularly important for some studies, such as those addressing host-pathogen interactions in C. elegans, since certain microbes do not efficiently infect worms in liquid culture.

We show how the method can be used to quantify the importance of genes in anti-fungal innate immunity in C. elegans. In this case, the approach relies on the use of a transgenic strain carrying an epidermal infection-inducible fluorescent reporter gene, with GFP under the control of the promoter of the antimicrobial peptide gene nlp 29 and a red fluorescent reporter that is expressed constitutively in the epidermis. The latter provides an internal control for the functional integrity of the epidermis and nonspecific transgene silencing9. When control worms are infected by the fungus they fluoresce green. Knocking down by RNAi a gene required for nlp 29 expression results in diminished fluorescence after infection. Currently, this protocol allows more than 3,000 RNAi clones to be tested and analyzed per week, opening the possibility of screening the entire genome in less than 2 months.

RNA interference (RNAi) has become a powerful genetic approach to systematically dissect gene function on a genome-wide scale. Owing to the penetrance and efficiency of RNAi in invertebrates, model organisms such as Drosophila melanogaster and Caenorhabditis elegans have contributed significantly to the identification of novel components of diverse biological pathways, ranging from early development to fat storage and aging. For the correct assessment of phenotypes, a key issue remains the stringent quality control of long double-stranded RNAs (dsRNA) to calculate potential off-target effects that may obscure the phenotypic data. We here describe a web-based tool to evaluate and design optimized dsRNA constructs. Moreover, the application also gives access to published predesigned dsRNAs. The E-RNAi web application is available at .

Background

High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention.

Results

We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene.

Conclusions

RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.

The phenomenon that is known as RNA mediated interference (RNAi) was first observed in the nematode C. elegans. The application of RNAi has now been widely disseminated and the mechanisms underlying the pathway have been uncovered using both genetics and biochemistry. In the worm, it has been demonstrated that RNAi is easily adapted to high throughput analysis and screening protocols. Hence, given the availability of whole genome sequences, RNAi has been used extensively as a tool for annotating gene function. Genetic screens performed with C. elegans have also led to the identification of genes that are essential for RNAi or that modulate the RNAi process. The identification of such genes has made it possible to manipulate and enhance the RNAi response. Moreover, many of the genes identified in C. elegans have been conserved in other organisms. Thus, opportunities are available for researchers to take advantage of the insights gained from the worm and apply them to their own systems in order to improve the efficiency and potency of the RNAi response.

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites.

Background

Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ß-catenin pathway.

Methodology/Principal Findings

In pursuit of such insights, we performed a genome-wide RNAi screen in Drosophila cells expressing an activated form of LRP6 and a β-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/β-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/β-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation.

Conclusions

These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/β-catenin pathway.

Background

The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies.

Results

We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA.

Conclusions

Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.

The completion of the genome sequencing for several organisms has created a great demand for genomic tools that can systematically analyze the growing wealth of data. In contrast to the classical reverse genetics approach of creating specific knockout cell lines or animals that is time-consuming and expensive, RNA-mediated interference (RNAi) has emerged as a fast, simple, and cost-effective technique for gene knockdown in large scale. Since its discovery as a gene silencing response to double-stranded RNA (dsRNA) with homology to endogenous genes in Caenorhabditis elegans (C elegans), RNAi technology has been adapted to various high-throughput screens (HTS) for genome-wide loss-of-function (LOF) analysis. Biochemical insights into the endogenous mechanism of RNAi have led to advances in RNAi methodology including RNAi molecule synthesis, delivery, and sequence design. In this article, we will briefly review these various RNAi library designs and discuss the benefits and drawbacks of each library strategy.

Background

The analysis of high-throughput screening data sets is an expanding field in bioinformatics. High-throughput screens by RNAi generate large primary data sets which need to be analyzed and annotated to identify relevant phenotypic hits. Large-scale RNAi screens are frequently used to identify novel factors that influence a broad range of cellular processes, including signaling pathway activity, cell proliferation, and host cell infection. Here, we present a web-based application utility for the end-to-end analysis of large cell-based screening experiments by cellHTS2.

Results

The software guides the user through the configuration steps that are required for the analysis of single or multi-channel experiments. The web-application provides options for various standardization and normalization methods, annotation of data sets and a comprehensive HTML report of the screening data analysis, including a ranked hit list. Sessions can be saved and restored for later re-analysis. The web frontend for the cellHTS2 R/Bioconductor package interacts with it through an R-server implementation that enables highly parallel analysis of screening data sets. web cellHTS2 further provides a file import and configuration module for common file formats.

Conclusions

The implemented web-application facilitates the analysis of high-throughput data sets and provides a user-friendly interface. web cellHTS2 is accessible online at http://web-cellHTS2.dkfz.de. A standalone version as a virtual appliance and source code for platforms supporting Java 1.5.0 can be downloaded from the web cellHTS2 page. web cellHTS2 is freely distributed under GPL.

Background

RNAi is a prominent tool for the identification of novel regulatory elements within complex cellular pathways. In invertebrates, RNAi is a relatively straightforward process, where large double-stranded RNA molecules initiate sequence-specific transcript destruction in target cells. In contrast, RNAi in mammalian cell culture assays requires the delivery of short interfering RNA duplexes to target cells. Due to concerns over off-target phenotypes and extreme variability in duplex efficiency, investigators typically deliver and analyze multiple duplexes per target. Currently, duplexes are delivered and analyzed either individually or as a pool of several independent duplexes. A choice between experiments based on siRNA pools or multiple individual duplexes has considerable implications for throughput, reagent requirements and data analysis in genome-wide surveys, yet there are relatively few data that directly compare the efficiency of the two approaches.

Methodology/Principal Findings

To address this critical issue, we conducted a direct comparison of siRNA pools and multiple single siRNAs that target all human phosphatases in a robust functional assay. We determined the frequency with which both approaches uncover loss-of-function phenotypes and compared the phenotypic severity for siRNA pools and the constituent individual duplexes.

Conclusions/Significance

Our survey indicates that screens with siRNA pools have several significant advantages over identical screens with the corresponding individual siRNA duplexes. Of note, we frequently observed greater phenotypic penetrance for siRNA pools than for the parental individual duplexes. Thus, our data indicate that experiments with siRNA pools have a greater likelihood of generating loss-of-function phenotypes than individual siRNA duplexes.

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.

The screen suggested functions for 393 genes for which no RNAi-mediated phenotype was known. The comparison with similar screens in worms has general implications for RNAi experiments

Gene silencing by transient or stable RNA-interference (RNAi) is used for the study of apoptosis with an assumption that apoptotic events will not influence RNAi. However, we recently reported that stable RNAi, i.e., a permanent gene-knockdown mediated by shRNA-generating DNA vectors that are integrated in the genome, fails rapidly after induction of apoptosis due to caspase-3-mediated cleavage and inactivation of the endoribonuclease Dicer-1 that is required for conversion of shRNA to siRNA. Since apoptosis studies also increasingly employ transient RNAi models in which apoptosis is induced immediately after a gene is temporarily knocked down within a few days of transfection with RNAi-inducing agents, we examined the impact of apoptosis on various models of transient RNAi. We report here that unlike the stable RNAi, all forms of transient RNAi, whether Dicer-1-independent (by 21mer dsRNA) or Dicer-1-dependent (by 27mer dsRNA or shRNA-generating DNA vector), whether for an exogenous gene GFP or an endogenous gene poly(ADP-ribose) polymerase-1, do not fail for 2–3 days after onset of apoptosis. Our results reflect the differences in dynamics of achieving and maintaining RNAi during the early phase after transfection in the transient RNAi model and the late steady-state phase of gene-knockdown in stable RNAi model. Our results also sound a cautionary note that RNAi status should be frequently validated in the studies involving apoptosis and that while stable RNAi can be safely used for the study of early apoptotic events, transient RNAi is more suitable for the study of both early and late apoptotic events.

RNA interference (RNAi) pathways are widespread in metaozoans but the genes required show variable occurrence or activity in eukaryotic microbes, including many pathogens. While some Leishmania lack RNAi activity and Argonaute or Dicer genes, we show that Leishmania braziliensis and other species within the Leishmania subgenus Viannia elaborate active RNAi machinery. Strong attenuation of expression from a variety of reporter and endogenous genes was seen. As expected, RNAi knockdowns of the sole Argonaute gene implicated this protein in RNAi. The potential for functional genetics was established by testing RNAi knockdown lines lacking the paraflagellar rod, a key component of the parasite flagellum. This sets the stage for the systematic manipulation of gene expression through RNAi in these predominantly diploid asexual organisms, and may also allow selective RNAi-based chemotherapy. Functional evolutionary surveys of RNAi genes established that RNAi activity was lost after the separation of the Leishmania subgenus Viannia from the remaining Leishmania species, a divergence associated with profound changes in the parasite infectious cycle and virulence. The genus Leishmania therefore offers an accessible system for testing hypothesis about forces that may select for the loss of RNAi during evolution, such as invasion by viruses, changes in genome plasticity mediated by transposable elements and gene amplification (including those mediating drug resistance), and/or alterations in parasite virulence.

Author Summary

RNAi interference pathways play fundamental roles in eukaryotes and provide important methods for the analysis of gene function. Occasionally RNAi has been lost, precluding its use as a tool, as well as raising the question of what forces could lead to loss of such a key pathway. Genomic and functional studies previously showed that within trypanosomatids protozoans RNAi was absent in both Leishmania major and Trypanosoma cruzi. The genome of L. braziliensis, a member of the early diverging Leishmania subgenus Viannia, retained key genes required for RNAi such as an Argonaute. We demonstrated that in fact L. braziliensis shows strong RNAi activity with reporter and endogenous genes affecting flagellar function. These data suggest that RNAi may be productively applied for functional genomic studies in L. braziliensis. We mapped the evolutionary point at which RNAi was lost in lineage leading to Leishmania and Crithidia, and establish that RNAi must have been lost at least twice in the trypanosomatids, once on the lineage leading to T. cruzi and independently following the divergence of the Viannia subgenus from other Leishmania species. Lastly, we discuss hypotheses concerning the forces leading to the loss of RNAi in Leishmania evolution, including viral invasion, increased genome plasticity, and altered virulence.