Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes (“hits”) and nonsignificant genes (“nonhits”) of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G1, or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G1/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-d-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.
Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases.
The Flavivirus genus includes several emergent and reemergent viruses, such as dengue and yellow fever viruses, which cause severe diseases in humans for which there is no approved treatment. Flaviviruses are transmitted to humans by arthropods and they rely on scores of vertebrate and invertebrate factors to replicate in these disparate hosts. Identifying the host factors involved in viral propagation is critical to understanding the molecular mechanisms of infection and the development of new therapeutics. To identify human host factors required for yellow fever virus propagation, we completed two genome-scale siRNA screens. Among the candidates discovered were the G protein-coupled receptor kinases GRK2 and GRK4. We focused on the protein GRK2, a kinase first identified for its role in cellular signal transduction. We found that GRK2 was a host factor needed for productive infection by yellow fever, dengue and hepatitis C viruses and was required for both viral entry and efficient replication of the viral genome. GRKs, which are considered druggable, may be used as targets to develop broadspectrum anti-flavivirals.
The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.
Influenza virus A (IVA) infection is responsible for recent death worldwide. Hence, there is a need to develop therapeutic agents against the virus. We describe the
prediction of short interfering RNA (siRNA) as potential therapeutic molecules for the HA (Haemagglutinin) and NA (Neuraminidase) genes. We screened 90,522
siRNA candidates for HA and 13,576 for NA and selected 1006 and 1307 candidates for HA and NA, respectively based on the proportion of viral sequences that
are targeted by the corresponding siRNA, with complete matches. Further short listing to select siRNA with no off-target hits, fulfilling all the guidelines
mentioned in approach, provided us 13 siRNAs for haemagglutinin and 13 siRNAs for neuraminidase. The approach of finding siRNA using multiple sequence
alignments of amino acid sequences has led to the identification of five conserved amino acid sequences, three in hemagglutinin i.e. RGLFGAIAGFIE,
YNAELLV and AIAGFIE and two in neuraminidase i.e. RTQSEC and EECSYP which on reveres translation provided siRNA sequences as potential therapeutic
candidates. The approaches used during this study have enabled us to identify potentially therapeutic siRNAs against divergent IVA strains.
Influenza virus A; Sequence analysis; siRNA; Hemagglutinin; Neuraminidase
RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53+/− matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity ∼4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.
Large-scale RNA interference (RNAi) experiments, especially the ones based on short-interfering RNA (siRNA) technology became increasingly popular over the past years. For such knock-down/screening purposes, different companies offer sets of oligos/reagents targeting the whole genome or a subset of it for various organisms. Obviously, the sequence (and structure) of the corresponding oligos is a key factor in obtaining reliable results in these large-scale studies and the companies use a variety of (often not fully public) algorithms to design them. Nevertheless, as the genome annotations are still continuously changing, oligos may become obsolete, so siRNA reagents should be periodically re-annotated according to the latest version of the sequence database (which of course has serious consequences also on the interpretation of the screening results). In our article, we would like to introduce a new software/database tool, the RNAiAtlas. It has been created for exploration, analysis and distribution of large scale RNAi libraries (currently limited to the human genome) with their latest annotation (including former history) but in addition it contains also specific on-target analysis results (design quality, side effects, off-targets).
RNA interference (RNAi) has been seen as a revolution in functional genomics and system biology. Genome-wide RNAi research relies on the development of RNAi high-throughput screening (HTS) assays. One of the most fundamental challenges in RNAi HTS is to glean biological significance from mounds of data, which relies on the development of effective analytic methods for selecting effective small interfering RNAs (siRNAs).
Based on a recently proposed parameter, strictly standardized mean difference (SSMD), I propose an analytic method for genome-wide screens of effective siRNAs through assessing and testing the size of siRNA effects. Central to this method is the capability of SSMD in quantifying siRNA effects. This method has relied on normal approximation, which works only in the primary screens but not in the confirmatory screens. In this paper, I explore the non-central t-distribution property of SSMD estimates and use this property to extend the SSMD-based method so that it works effectively in either primary or confirmatory screens as well as in any HTS screens with or without replicates. The SSMD-based method maintains a balanced control of false positives and false negatives.
The central interest in genome-wide RNAi research is the selection of effective siRNAs which relies on the development of analytic methods to measure the size of siRNA effects. The new analytic method for hit selection provided in this paper offers a good analytic tool for selecting effective siRNAs, better than current analytic methods, and thus may have broad utility in genome-wide RNAi research.
RNA interference (RNAi) has opened promising avenues to better understand gene function. Though many RNAi screens report on the identification of genes, very few, if any, have been further studied and validated. Data discrepancy is emerging as one of RNAi main pitfalls. We reasoned that a systematic analysis of lethality-based screens, since they score for cell death, would examine the extent of hit discordance at inter-screen level. To this end, we developed a methodology for literature mining and overlap analysis of several screens using both siRNA and shRNA flavors, and obtained 64 gene lists censoring an initial list of 7,430 nominated genes. We further performed a comparative analysis first at a global level followed by hit re-assessment under much more stringent conditions. To our surprise, none of the hits overlapped across the board even for PLK1, which emerged as a strong candidate in siRNA screens; but only marginally in the shRNA ones. Furthermore, EIF5B emerges as the most common hit only in the shRNA screens. A highly unusual and unprecedented result was the observation that 5,269 out of 6,664 nominated genes (~80%) in the shRNA screens were exclusive to the pooled format, raising concerns as to the merits of pooled screens which qualify hits based on relative depletions, possibly due to multiple integrations per cell, data deconvolution or inaccuracies in intracellular processing causing off-target effects. Without golden standards in place, we would encourage the community to pay more attention to RNAi screening data analysis practices, bearing in mind that it is combinatorial in nature and one active siRNA duplex or shRNA hairpin per gene does not suffice credible hit nomination. Finally, we also would like to caution interpretation of pooled shRNA screening outcomes.
RNAi; shRNA; siRNA; Gene; screening; bioinformatics; analysis; overlap; lethality; essential; PLK1
Small interfering RNAs (siRNAs) have become a ubiquitous experimental tool for down-regulating mRNAs. Unfortunately, off-target effects are a significant source of false positives in siRNA experiments and an effective control for them has not previously been identified. We introduce two methods of mismatched siRNA design for negative controls based on changing bases in the middle of the siRNA to their complement bases. To test these controls, a test set of 20 highly active siRNAs (10 true positives and 10 false positives) was identified from a genome-wide screen performed in a cell-line expressing a simple, constitutively expressed luciferase reporter. Three controls were then synthesized for each of these 20 siRNAs, the first two using the proposed mismatch design methods and the third being a simple random permutation of the sequence (scrambled siRNA). When tested in the original assay, the scrambled siRNAs showed significantly reduced activity in comparison to the original siRNAs, regardless of whether they had been identified as true or false positives, indicating that they have little utility as experimental controls. In contrast, one of the proposed mismatch design methods, dubbed C911 because bases 9 through 11 of the siRNA are replaced with their complement, was able to completely distinguish between the two groups. False positives due to off-target effects maintained most of their activity when the C911 mismatch control was tested, whereas true positives whose phenotype was due to on-target effects lost most or all of their activity when the C911 mismatch was tested. The ability of control siRNAs to distinguish between true and false positives, if widely adopted, could reduce erroneous results being reported in the literature and save research dollars spent on expensive follow-up experiments.
In order to identify novel proviral host factors involved in human immunodeficiency virus (HIV) infection, we performed a screen of a small interfering RNA (siRNA) library targeting 5,000 genes with the highest potential for being targets for therapeutics. Many siRNAs in the library against known host factors, such as TSG101, furin, and CXCR4, were identified as inhibitors by the screen and thus served as internal validation. In addition, many novel factors whose knockdown inhibited infection were identified, including Pak3, a member of the serine/threonine group I PAK kinases. The HIV accessory factor Nef has been shown to associate with a PAK kinase, leading to enhanced viral production; however, the exact identity of the kinase has remained controversial. Prompted by the Pak3 screen hit, we further investigated the involvement of group I PAK kinases in HIV using siRNA. Contrary to the current literature, Pak1 depletion strongly inhibited HIV infection in multiple cell systems and decreased levels of integrated provirus, while Pak2 depletion showed no effect. Overexpression of a constitutively active Pak1 mutant also enhanced HIV infection, further supporting its role as the dominant PAK involved.
Toxoplasma gondii critically relies on cell invasion as a survival strategy to evade immune clearance during infection. Although it was widely thought that Toxoplasma entry is parasite directed and that the host cell is largely a passive victim, recent studies have suggested that host components such as microfilaments and microtubules indeed contribute to entry. Hence to identify additional host factors, we performed a high-throughput siRNA screen of a human siRNA library targeting druggable proteins using a novel inducible luciferase based invasion assay. The top 100 hits from the primary screen that showed the strongest decreases in invasion were subjected to confirmation by secondary screening, revealing 24 proteins that are potentially involved in Toxoplasma entry into host cells. Interestingly, 6 of the hits appear to affect parasite invasion by modifying host cell actin dynamics, resulting in increased deposition of F-actin at the periphery of the cell. These findings support the emerging notion that host actin dynamics are important for Toxoplasma invasion along with identifying several novel host factors that potentially participate in parasite entry.
Technological advances in both siRNA (small interfering RNA) and whole genome sequencing have demonstrated great potential in translating genetic information into siRNA-based drugs to halt the synthesis of most disease-causing proteins. Despite its powerful promises as a drug, siRNA requires a sophisticated delivery vehicle because of its rapid degradation in the circulation, inefficient accumulation in target tissues and inability to cross cell membranes to access the cytoplasm where it functions. Lipid nanoparticle (LNP) containing ionizable amino lipids is the leading delivery technology for siRNA, with five products in clinical trials and more in the pipeline. Here, we focus on the technological advances behind these potent systems for siRNA-mediated gene silencing.
lipid nanoparticle; siRNA; ionizable amino lipid; pKa; PEG lipid; targeting
Some barriers to DNA lipofection are well characterized; however, there is as yet no method of finding unknown pathways that impact the process. A druggable genome small-interfering RNA (siRNA) screen against 5,520 genes was tested for its effect on lipofection of human aortic endothelial cells (HAECs). We found 130 gene targets which, when silenced by pooled siRNAs (three siRNAs per gene), resulted in enhanced luminescence after lipofection (86 gene targets showed reduced expression). In confirmation tests with single siRNAs, 18 of the 130 hits showed enhanced lipofection with two or more individual siRNAs in the absence of cytotoxicity. Of these confirmed gene targets, we identified five leading candidates, two of which are isoforms of the regulatory subunit of protein phosphatase 2A (PP2A). The best candidate siRNA targeted the PPP2R2C gene and produced a 65% increase in luminescence from lipofection, with a quantitative PCR–validated knockdown of ~76%. Flow cytometric analysis confirmed that the silencing of the PPP2R2C gene resulted in an improvement of 10% in transfection efficiency, thereby demonstrating an increase in the number of transfected cells. These results show that an RNA interference (RNAi) high-throughput screen (HTS) can be applied to nonviral gene transfer. We have also demonstrated that siRNAs can be co-delivered with lipofected DNA to increase the transfection efficiency in vitro.
RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer.
We assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-β pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-β receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 expression.
Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-β receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to the methods to reduce or predict RNAi off-target effects would be beneficial.
DNA microarrays allow many simultaneous parallel measurements; and transcriptional profiling has provided scientists with a wealth of information. The same technology can be used to build protein arrays which hold similar promise. We describe here an approach using protein arrays to screen cells for gene knockdowns. Small interfering RNAs (siRNA) can be used to degrade mRNA levels of a specific gene, thereby reducing the corresponding protein levels within cells. The use of siRNAs has many implications in functional genomics and proteomics as well as therapeutics, as researchers use this tool to identify proteins involved in specific pathways. We demonstrate the use of microarrays as a screening method to identify cells that have been treated successfully with a beta-actin siRNA.
We have silenced the β-actin gene in HeLa cells using siLentMer Dicer-Substrate siRNA Duplexes, and confirmed the knockdown with a combination of 2-D gel analysis, western blotting and quantitative PCR (qPCR). An increase in the level of phosphorylated cofilin was also detected (Liu, N., et al. 2006 in press).
We now demonstrate the use of reverse phase protein microarrays to screen for these proteins. These arrays enable higher throughput than traditional protein detection, and add multiplexing capabilities. Arrays were produced on the benchtop with the BioOdyssey Calligrapher miniarrayer. Antibodies against β-actin and phosphocofilin were tested for specificity by western blots. In one experiment, arrays were processed to monitor the concentration of β-actin in cells, with a standard curve of purified human actin printed on the grids. In another experiment, changes in phosphorylation levels of cofilin with an antibody specific for phosphocofilin were detected. We also demonstrate that these printed arrays can be screened using antibodies either singly or in pairs. Finally, the microarray results were validated using qPCR.
The cytoplasmic receptor NOD2 (nucleotide-binding oligomerization domain 2) senses peptidoglycan fragments and triggers host defense pathways that lead to inflammatory immune responses. Dysregulation of NOD2 signaling is associated with inflammatory diseases, such as Crohn’s disease and Blau syndrome. We used a genome-wide, small interfering RNA (siRNA) screen to identify regulators of the NOD2 signaling pathway. Several genes associated with Crohn’s disease risk were identified in the screen, supporting a role for NOD2 and nuclear factor κB (NF-κB) pathways in the pathogenesis of Crohn’s disease. A comparison of hits from this screen with other “omics” data sets revealed interconnected networks of genes implicated in NF-κB signaling. Secondary assays, including the measurement of interleukin-8 secretion, served to validate many of the regulators. Knockdown of putative regulators in HEK293 cells followed by stimulation with tumor necrosis factor α revealed that most of the genes identified were general regulators of NF-κB signaling. Overall, the genes identified here provide a resource to facilitate the elucidation of the molecular mechanisms that regulate NOD2- and NF-κB–mediated inflammation.
Application of siRNA in high-throughput fashion is still in its early phase although the principle has been established for three years. In this review, we outline the different vector-based siRNA delivery platforms as well as resources that are becoming available for high-throughput applications, and some initial outcomes of vector siRNA high-throughput screening efforts using vector encoded siRNA. It is expected that further improvement of the siRNA technology and availability of the siRNA resources will help to materialize the potential of siRNA for functional genomics and drug target validation.
siRNA; RNAi; vectors; gene silencing; siRNA delivery
Coxiella burnetii is an intracellular pathogen that replicates within a lysosome-like vacuole. A Dot/Icm type IVB secretion system is used by C. burnetii to translocate effector proteins into the host cytosol that likely modulate host factor function. To identify host determinants required for C. burnetii intracellular growth, a genome-wide screen was performed using gene silencing by small interfering RNA (siRNA). Replication of C. burnetii was measured by immunofluorescence microscopy in siRNA-transfected HeLa cells. Newly identified host factors included components of the retromer complex, which mediates cargo cycling between the endocytic pathway and the Golgi apparatus. Reducing the levels of the retromer cargo-adapter VPS26-VPS29-VPS35 complex or retromer-associated sorting nexins abrogated C. burnetii replication. Several genes, when silenced, resulted in enlarged vacuoles or an increased number of vacuoles within C. burnetii-infected cells. Silencing of the STX17 gene encoding syntaxin-17 resulted in a striking defect in homotypic fusion of vacuoles containing C. burnetii, suggesting a role for syntaxin-17 in regulating this process. Lastly, silencing host genes needed for C. burnetii replication correlated with defects in the translocation of Dot/Icm effectors, whereas, silencing of genes that affected vacuole morphology, but did not impact replication, did not affect Dot/Icm translocation. These data demonstrate that C. burnetii vacuole maturation is important for creating a niche that permits Dot/Icm function. Thus, genome-wide screening has revealed host determinants involved in sequential events that occur during C. burnetii infection as defined by bacterial uptake, vacuole transport and acidification, activation of the Dot/Icm system, homotypic fusion of vacuoles, and intracellular replication.
Q fever in humans is caused by the bacterium Coxiella burnetii. Infection with C. burnetii is marked by its unique ability to replicate within a large vacuolar compartment inside cells that resembles the harsh, acidic environment of a lysosome. Central to its pathogenesis is the delivery of bacterial effector proteins into the host cell cytosol by a Dot/Icm type IVB secretion system. These proteins can interact with and manipulate host factors, thereby leading to creation and maintenance of the vacuole that the bacteria grow within. Using high-throughput genome-wide screening in human cells, we identified host factors important for several facets of C. burnetii infection, including vacuole transport and membrane fusion events that promote vacuole expansion. In addition, we show that maturation of the C. burnetii vacuole is necessary for creating an environment permissive for the Dot/Icm delivery of bacterial effector proteins into the host cytosol.
Small interfering RNA (siRNA)-based technology is playing a pivotal role in understanding gene function. Huge siRNA libraries coupled to high-content screening are being applied to decipher molecular circuitries, as well as to identify novel therapeutic targets. Further, the technology is finding its way towards therapeutic applications. In the midst of all this excitement, the siRNA technology is faced with challenges, arising mostly from siRNAs being a nucleic acid molecule, and also from the baggage it inherits from the RNA interference (RNAi) pathway, which is critical to the function of siRNAs.
RNAi; siRNA; microRNA; gene silencing; off-target effects; siRNA design; siRNA delivery; shRNA expression; immune activation
Small interfering RNAs (siRNAs) have become an important tool in cell and molecular biology. Reliable design of siRNA molecules is essential for the needs of large functional genomics projects.
To improve the design of efficient siRNA molecules, we performed a comparative, thermodynamic and correlation analysis on a heterogeneous set of 653 siRNAs collected from the literature. We used this training set to select siRNA features and optimize computational models. We identified 18 parameters that correlate significantly with silencing efficiency. Some of these parameters characterize only the siRNA sequence, while others involve the whole mRNA. Most importantly, we derived an siRNA position-dependent consensus, and optimized the free-energy difference of the 5' and 3' terminal dinucleotides of the siRNA antisense strand. The position-dependent consensus is based on correlation and t-test analyses of the training set, and accounts for both significantly preferred and avoided nucleotides in all sequence positions. On the training set, the two parameters' correlation with silencing efficiency was 0.5 and 0.36, respectively. Among other features, a dinucleotide content index and the frequency of potential targets for siRNA in the mRNA added predictive power to our model (R = 0.55). We showed that our model is effective for predicting the efficiency of siRNAs at different concentrations.
We optimized a neural network model on our training set using three parameters characterizing the siRNA sequence, and predicted efficiencies for the test siRNA dataset recently published by Novartis. On this validation set, the correlation coefficient between predicted and observed efficiency was 0.75. Using the same model, we performed a transcriptome-wide analysis of optimal siRNA targets for 22,600 human mRNAs.
We demonstrated that the properties of the siRNAs themselves are essential for efficient RNA interference. The 5' ends of antisense strands of efficient siRNAs are U-rich and possess a content similarity to the pyrimidine-rich oligonucleotides interacting with the polypurine RNA tracks that are recognized by RNase H. The advantage of our method over similar methods is the small number of parameters. As a result, our method requires a much smaller training set to produce consistent results. Other mRNA features, though expensive to compute, can slightly improve our model.
Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa® Nucleofector® 96-well Shuttle® System for siRNA screening in primary cells. Lonza's Clonetics® HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME® siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector® 96-well Shuttle® System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.
Nucleofection; RNAi; siRNA; primary cell; screening; transfection; HUVEC
Clathrin-mediated endocytosis is essential for a wide range of cellular functions. We used a multi-step siRNA-based screening strategy to identify regulators of the first step in clathrin-mediated endocytosis, formation of clathrin-coated vesicles (CCVs) at the plasma membrane. A primary genome-wide screen identified 334 hits that caused accumulation of CCV cargo on the cell surface. A secondary screen identified 92 hits that inhibited cargo uptake and/or altered the morphology of clathrin-coated structures. The hits include components of four functional complexes: coat proteins, V-ATPase subunits, spliceosome-associated proteins, and acetyltransferase subunits. Electron microscopy revealed that V-ATPase depletion caused the cell to form aberrant non-constricted clathrin-coated structures at the plasma membrane. The V-ATPase knockdown phenotype was rescued by addition of exogenous cholesterol, indicating that the knockdown blocks clathrin-mediated endocytosis by preventing cholesterol from recycling from endosomes back to the plasma membrane.
The design of small interfering RNA (siRNA) is a multi factorial problem that has gained the attention of many researchers in the area of therapeutic and functional genomics. MysiRNA score was previously introduced that improves the correlation of siRNA activity prediction considering state of the art algorithms. In this paper, a new program, MysiRNA-Designer, is described which integrates several factors in an automated work-flow considering mRNA transcripts variations, siRNA and mRNA target accessibility, and both near-perfect and partial off-target matches. It also features the MysiRNA score, a highly ranked correlated siRNA efficacy prediction score for ranking the designed siRNAs, in addition to top scoring models Biopredsi, DISR, Thermocomposition21 and i-Score, and integrates them in a unique siRNA score-filtration technique. This multi-score filtration layer filters siRNA that passes the 90% thresholds calculated from experimental dataset features. MysiRNA-Designer takes an accession, finds conserved regions among its transcript space, finds accessible regions within the mRNA, designs all possible siRNAs for these regions, filters them based on multi-scores thresholds, and then performs SNP and off-target filtration. These strict selection criteria were tested against human genes in which at least one active siRNA was designed from 95.7% of total genes. In addition, when tested against an experimental dataset, MysiRNA-Designer was found capable of rejecting 98% of the false positive siRNAs, showing superiority over three state of the art siRNA design programs. MysiRNA is a freely accessible (Microsoft Windows based) desktop application that can be used to design siRNA with a high accuracy and specificity. We believe that MysiRNA-Designer has the potential to play an important role in this area.
Replicative senescence forms a major barrier to tumor progression. Cancer cells bypass this by using one of the two known telomere maintenance mechanisms: telomerase or the recombination-based alternative lengthening of telomeres (ALT) mechanism. The molecular details of ALT are currently poorly understood. We have previously shown that telomerase is actively repressed through complex networks of kinase, gene expression, and chromatin regulation. In this study, we aimed to gain further understanding of the role of kinases in the regulation of telomerase expression in ALT cells. Using a whole human kinome small interfering RNA (siRNA) screen, we highlighted 106 kinases whose expression is linked to human telomerase reverse transcriptase (hTERT) promoter activity. Network modeling of transcriptional regulation implicated c-Myc as a key regulator of the 106 kinase hits. Given our previous observations of lower c-Myc activity in ALT cells, we further explored its potential to regulate telomerase expression in ALT. We found increased c-Myc binding at the hTERT promoter in telomerase-positive compared with ALT cells, although no expression differences in c-Myc, Mad, or Max were observed between ALT and telomerase-positive cells that could explain decreased c-Myc activity in ALT. Instead, we found increased expression of the c-Myc competitive inhibitor TCEAL7 in ALT cells and tumors and that alteration of TCEAL7 expression levels in ALT and telomerase-positive cells affects hTERT expression. Lower c-Myc activity in ALT may therefore be obtained through TCEAL7 regulation. Thus, TCEAL7 may present an interesting novel target for cancer therapy, which warrants further investigation.
Eukaryotic cells express a wide variety of endogenous small regulatory RNAs that regulate heterochromatin formation, developmental timing, defense against parasitic nucleic acids, and genome rearrangement. Many small regulatory RNAs are thought to function in nuclei 1-2. For instance, in plants and fungi siRNAs associate with nascent transcripts and direct chromatin and/or DNA modifications 1-2. To further understand the biological roles of small regulatory RNAs, we conducted a genetic screen to identify factors required for RNA interference (RNAi) in C. elegans nuclei 3. Here we show that nrde-2 encodes an evolutionarily conserved protein that is required for small interfering (si)RNA-mediated silencing in nuclei. NRDE-2 associates with the Argonaute protein NRDE-3 within nuclei and is recruited by NRDE-3/siRNA complexes to nascent transcripts that have been targeted by RNAi. We find that nuclear-localized siRNAs direct a NRDE-2-dependent silencing of pre-mRNAs 3’ to sites of RNAi, a NRDE-2-dependent accumulation of RNA Polymerase (RNAP) II at genomic loci targeted by RNAi, and NRDE-2-dependent decreases in RNAP II occupancy and RNAP II transcriptional activity 3’ to sites of RNAi. These results define NRDE-2 as a component of the nuclear RNAi machinery and demonstrate that metazoan siRNAs can silence nuclear-localized RNAs co-transcriptionally. In addition, these results establish a novel mode of RNAP II regulation; siRNA-directed recruitment of NRDE factors that inhibit RNAP II during the elongation phase of transcription.