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1.  Replication of Many Human Viruses Is Refractory to Inhibition by Endogenous Cellular MicroRNAs 
Journal of Virology  2014;88(14):8065-8076.
The issue of whether viruses are subject to restriction by endogenous microRNAs (miRNAs) and/or by virus-induced small interfering RNAs (siRNAs) in infected human somatic cells has been controversial. Here, we address this question in two ways. First, using deep sequencing, we demonstrate that infection of human cells by the RNA virus dengue virus (DENV) or West Nile virus (WNV) does not result in the production of any virus-derived siRNAs or viral miRNAs. Second, to more globally assess the potential of small regulatory RNAs to inhibit virus replication, we used gene editing to derive human cell lines that lack a functional Dicer enzyme and that therefore are unable to produce miRNAs or siRNAs. Infection of these cells with a wide range of viruses, including DENV, WNV, yellow fever virus, Sindbis virus, Venezuelan equine encephalitis virus, measles virus, influenza A virus, reovirus, vesicular stomatitis virus, human immunodeficiency virus type 1, or herpes simplex virus 1 (HSV-1), failed to reveal any enhancement in the replication of any of these viruses, although HSV-1, which encodes at least eight Dicer-dependent viral miRNAs, did replicate somewhat more slowly in the absence of Dicer. We conclude that most, and perhaps all, human viruses have evolved to be resistant to inhibition by endogenous human miRNAs during productive replication and that dependence on a cellular miRNA, as seen with hepatitis C virus, is rare. How viruses have evolved to avoid inhibition by endogenous cellular miRNAs, which are generally highly conserved during metazoan evolution, remains to be determined.
IMPORTANCE Eukaryotic cells express a wide range of small regulatory RNAs, including miRNAs, that have the potential to inhibit the expression of mRNAs that show sequence complementarity. Indeed, previous work has suggested that endogenous miRNAs have the potential to inhibit viral gene expression and replication. Here, we demonstrate that the replication of a wide range of pathogenic viruses is not enhanced in human cells engineered to be unable to produce miRNAs, indicating that viruses have evolved to be resistant to inhibition by miRNAs. This result is important, as it implies that manipulation of miRNA levels is not likely to prove useful in inhibiting virus replication. It also focuses attention on the question of how viruses have evolved to resist inhibition by miRNAs and whether virus mutants that have lost this resistance might prove useful, for example, in the development of attenuated virus vaccines.
PMCID: PMC4097787  PMID: 24807715
2.  Biologic and Clinical Significance of Androgen Receptor Variants in Castration Resistant Prostate Cancer 
Endocrine-related cancer  2014;21(4):T87-T103.
As prostate cancer progresses to the lethal castration resistant and metastatic form, genetic and epigenetic adaptation, clonal selection, and evolution of the tumor microenvironment contribute to the emergence of unique biologic characteristics under the selective pressure of external stresses. These stresses include the therapies applied in the clinic or laboratory and the exposures of cancers to hormonal, paracrine, or autocrine stimuli in the context of the tumor micro- and macro-environment. The androgen receptor (AR) is a key gene involved in prostate cancer etiology and oncogenesis, including disease development, progression, response to initial hormonal therapies, and subsequent resistance to hormonal therapies. Alterations in the AR signaling pathway have been observed in certain selection contexts and contribute to the resistance to agents that target hormonal regulation of the AR, including standard androgen deprivation therapy (ADT), anti-androgens such as enzalutamide, and androgen synthesis inhibition with abiraterone acetate. One such resistance mechanism is the synthesis of constitutively active AR variants lacking the canonical ligand binding domain. This review focuses on the etiology, characterization, biologic properties, and emerging data contributing to the clinical characteristics of AR variants, and suggests approaches to full-length AR and AR variant biomarker validation, assessment, and systemic targeting in the clinic.
PMCID: PMC4277180  PMID: 24859991
Androgen receptor; splice variants; castration resistant prostate cancer; AR variants; metastasis; epithelial to mesenchymal transition
3.  Development of a method to isolate circulating tumor cells using mesenchymal-based capture 
Methods (San Diego, Calif.)  2013;64(2):10.1016/j.ymeth.2013.06.034.
Epithelial tumor cells can become mesenchymal cells and vice versa via phenotypic transitions, a process known as epithelial plasticity. We postulate that during the process of metastasis, circulating tumor cells (CTCs) lose their epithelial phenotype and acquire a mesenchymal phenotype that may not be sufficiently captured by existing epithelial-based CTC technologies. We report here on the development of a novel CTC capture method, based on the biology of epithelial plasticity, which isolates cells based on OB-cadherin cell surface expression. Using this mesenchymal-based assay, OB-cadherin cellular events are detectable in men with metastatic prostate cancer and are less common in healthy volunteers. This method may complement existing epithelial-based methods and may be particularly useful in patients with bone metastases.
PMCID: PMC3833983  PMID: 23845299
Circulating tumor cells; epithelial plasticity; epithelial-mesenchymal transition; OB-cadherin; osteomimicry; prostate cancer
4.  The role of epithelial plasticity in prostate cancer dissemination and treatment resistance 
Cancer metastasis reviews  2014;33(0):441-468.
Nearly 30,000 men die annually in the USA of prostate cancer, nearly uniformly from metastatic dissemination. Despite recent advances in hormonal, immunologic, bone-targeted, and cytotoxic chemotherapies, treatment resistance and further dissemination are inevitable in men with metastatic disease. Emerging data suggests that the phenomenon of epithelial plasticity, encompassing both reversible mesenchymal transitions and acquisition of stemness traits, may underlie this lethal biology of dissemination and treatment resistance. Understanding the molecular underpinnings of this cellular plasticity from preclinical models of prostate cancer and from biomarker studies of human metastatic prostate cancer has provided clues to novel therapeutic approaches that may delay or prevent metastatic disease and lethality over time. This review will discuss the preclinical and clinical evidence for epithelial plasticity in this rapidly changing field and relate this to clinical phenotype and resistance in prostate cancer while suggesting novel therapeutic approaches.
PMCID: PMC4230790  PMID: 24414193
Epithelial plasticity; Prostate cancer; Metastasis; Epithelial–mesenchymal transition; Dissemination; Stem cell
5.  G3BP1, G3BP2 and CAPRIN1 Are Required for Translation of Interferon Stimulated mRNAs and Are Targeted by a Dengue Virus Non-coding RNA 
PLoS Pathogens  2014;10(7):e1004242.
Viral RNA-host protein interactions are critical for replication of flaviviruses, a genus of positive-strand RNA viruses comprising major vector-borne human pathogens including dengue viruses (DENV). We examined three conserved host RNA-binding proteins (RBPs) G3BP1, G3BP2 and CAPRIN1 in dengue virus (DENV-2) infection and found them to be novel regulators of the interferon (IFN) response against DENV-2. The three RBPs were required for the accumulation of the protein products of several interferon stimulated genes (ISGs), and for efficient translation of PKR and IFITM2 mRNAs. This identifies G3BP1, G3BP2 and CAPRIN1 as novel regulators of the antiviral state. Their antiviral activity was antagonized by the abundant DENV-2 non-coding subgenomic flaviviral RNA (sfRNA), which bound to G3BP1, G3BP2 and CAPRIN1, inhibited their activity and lead to profound inhibition of ISG mRNA translation. This work describes a new and unexpected level of regulation for interferon stimulated gene expression and presents the first mechanism of action for an sfRNA as a molecular sponge of anti-viral effectors in human cells.
Author Summary
Dengue virus is the most prevalent arbovirus in the world and an increasingly significant public health problem. Development of vaccines and therapeutics has been slowed by poor understanding of viral pathogenesis. Especially, how the virus subverts the host interferon response, a powerful branch of the innate immune system remains the subject of debate and great interest. Dengue virus produces large quantities of a non-coding, highly structured viral RNA, termed sfRNA, whose function in viral replication is elusive but has been linked in related viruses to inhibition of the interferon response. Nonetheless the mechanisms involved are yet to be characterized. Here, we show that dengue virus 2 sfRNA targets and antagonizes a set of host RNA-binding proteins G3BP1, G3BP2 and CAPRIN1, to interfere with translation of antiviral interferon-stimulated mRNAs. This activity impairs establishment of the antiviral state, allowing the virus to replicate and evade the interferon response. While this particular mechanism was not conserved among other flaviviruses, we believe it is highly relevant for dengue virus 2 replication and pathogenesis. Taken together, our results highlight both new layers of complexity in the regulation of the innate immune response, as well as the diversity of strategies flaviviruses employ to counteract it.
PMCID: PMC4081823  PMID: 24992036
6.  Functional genomics approach for the identification of human host factors supporting dengue viral propagation 
Dengue virus (DENV) is endemic throughout tropical regions around the world and there are no approved treatments or anti-transmission agents currently available. Consequently, there exists an enormous unmet need to treat the human diseases caused by DENV and block viral transmission by the mosquito vector. RNAi screening represents an efficient method to expand the pool of known host factors that could become viable targets for treatments or provide rationale to consider available drugs as anti-DENV treatments. We developed a high throughput siRNA-based screening protocol that can identify human DENV host factors. The protocol herein describes the materials and the procedures necessary to screen a human cell line in order to identify genes which are either necessary for or restrict DENV propagation at any stage in the viral life cycle.
PMCID: PMC4075997  PMID: 24696344
RNA interference (RNAi); dengue virus; yellow fever virus; whole genome RNAi screening; whole genome siRNA screening; dengue virus host factors; flavivirus
7.  Human Genetic Variation and Yellow Fever Mortality during 19th Century U.S. Epidemics 
mBio  2014;5(3):e01253-14.
We calculated the incidence, mortality, and case fatality rates for Caucasians and non-Caucasians during 19th century yellow fever (YF) epidemics in the United States and determined statistical significance for differences in the rates in different populations. We evaluated nongenetic host factors, including socioeconomic, environmental, cultural, demographic, and acquired immunity status that could have influenced these differences. While differences in incidence rates were not significant between Caucasians and non-Caucasians, differences in mortality and case fatality rates were statistically significant for all epidemics tested (P < 0.01). Caucasians diagnosed with YF were 6.8 times more likely to succumb than non-Caucasians with the disease. No other major causes of death during the 19th century demonstrated a similar mortality skew toward Caucasians. Nongenetic host factors were examined and could not explain these large differences. We propose that the remarkably lower case mortality rates for individuals of non-Caucasian ancestry is the result of human genetic variation in loci encoding innate immune mediators.
Different degrees of severity of yellow fever have been observed across diverse populations, but this study is the first to demonstrate a statistically significant association between ancestry and the outcome of yellow fever (YF). With the global burden of mosquito-borne flaviviral infections, such as YF and dengue, on the rise, identifying and characterizing host factors could prove pivotal in the prevention of epidemics and the development of effective treatments.
PMCID: PMC4049105  PMID: 24895309
8.  Epstein-Barr Virus Induces Global Changes in Cellular mRNA Isoform Usage That Are Important for the Maintenance of Latency 
Journal of Virology  2013;87(22):12291-12301.
Oncogenic viruses promote cell proliferation through the dramatic reorganization of host transcriptomes. In addition to regulating mRNA abundance, changes in mRNA isoform usage can have a profound impact on the protein output of the transcriptome. Using Epstein-Barr virus (EBV) transformation of primary B cells, we have studied the ability of an oncogenic virus to alter the mRNA isoform profile of its host. Using the algorithm called SplicerEX with two complementary Affymetrix microarray platforms, we uncovered 433 mRNA isoform changes regulated by EBV during B-cell transformation. These changes were largely orthogonal with the 2,163 mRNA abundance changes observed during transformation, such that less than one-third of mRNAs changing at the level of isoform also changed in overall abundance. While we observed no preference for a mechanistic class of mRNA isoform change, we detected a significant shortening of 3′ untranslated regions and exclusion of cassette exons in EBV-transformed cells relative to uninfected B cells. Gene ontology analysis of the mRNA isoform changes revealed significant enrichment in nucleic acid binding proteins. We validated several of these isoform changes and were intrigued by those in two mRNAs encoding the proteins XBP1 and TCF4, which have both been shown to bind and activate the promoter of the major EBV lytic trans-activator BZLF1. Our studies indicate that EBV latent infection promotes the usage of mRNA isoforms of XBP1 and TCF4 that restrict BZLF1 activation. Therefore, characterization of global changes in mRNA isoform usage during EBV infection identifies a new mechanism for the maintenance of latent infection.
PMCID: PMC3807935  PMID: 24027308
9.  Targeting Host Factors to Treat West Nile and Dengue Viral Infections 
Viruses  2014;6(2):683-708.
West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus Flavivirus. At the current time, there are no approved prophylactics (e.g., vaccines) or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans.
PMCID: PMC3939478  PMID: 24517970
West Nile virus; dengue virus; host factor; drug targets
10.  Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication 
Retrovirology  2013;10:124.
Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo.
We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication.
Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication
PMCID: PMC3874760  PMID: 24165037
TCERG1; Transcription elongation; RNA polymerase II; Pausing
11.  Discovery of Insect and Human Dengue Virus Host Factors 
Nature  2009;458(7241):1047-1050.
Dengue fever (DF) is the most frequent arthropod-borne viral disease of humans, with almost half of the world's population at risk of infection1. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make DF a global public health threat1, 2. Given their compact genomes, dengue viruses (DENV 1-4) and other flaviviruses likely require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors have been identified3-10. To discover insect host factors required for DENV-2 propagation, we carried out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 dsRNA library. This screen identified 116 candidate dengue virus host factors (DVHFs) (Supplementary Fig. 1). While some were previously associated with flaviviruses (e.g., V-ATPases and alpha-glucosidases)3-5, 7, 9, 10, most DVHFs were newly implicated in DENV propagation. The dipteran DVHFs had eighty-two readily recognizable human homologues and, using a targeted siRNA screen, we showed that forty-two of these are human DVHFs. This indicates remarkable conservation of required factors between dipteran and human hosts. This work suggests novel approaches to control infection in the insect vector and the mammalian host.
PMCID: PMC3462662  PMID: 19396146
12.  G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication 
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.
Author Summary
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.
PMCID: PMC3441407  PMID: 23029581
13.  Circulating Tumor Cells from Patients with Advanced Prostate and Breast Cancer Display Both Epithelial and Mesenchymal Markers 
Molecular cancer research : MCR  2011;9(8):997-1007.
During cancer progression malignant cells undergo epithelial-mesenchymal transitions (EMTs) and mesenchymal-epithelial transitions (METs) as part of a broad invasion and metastasis program. We previously observed MET events among lung metastases in a preclinical model of prostate adenocarcinoma that suggested a relationship between epithelial plasticity and metastatic spread. We thus sought to translate these findings into clinical evidence by examining the existence of EMT in circulating tumor cells (CTCs) from patients with progressive metastatic solid tumors, with a focus on men with castration-resistant prostate cancer (CRPC) and women with metastatic breast cancer (BC). We show that the majority (>80%) of these CTCs in patients with metastatic CRPC co-express epithelial proteins such as EpCAM, CK, and E-cadherin, mesenchymal proteins, including vimentin, N-cadherin, and O-cadherin, and the stem cell marker CD133. Equally, we find that over 75% of CTCs from women with metastatic BC co-express cytokeratin, vimentin, and N-cadherin. The existence and high frequency of these CTCs co-expressing epithelial, mesenchymal, and stem-cell markers in patients with progressive metastases has important implications for the application and interpretation of approved methods to detect CTCs.
PMCID: PMC3157566  PMID: 21665936
metastatic castration resistant prostate cancer (mCRPC); epithelial mesenchymal transition (EMT); mesenchymal epithelial transition (MET); circulating tumor cells (CTCs); metastatic breast cancer; N-Cadherin; CD133; O-Cadherin; EpCAM
15.  Quantitative mass spectrometry of DENV-2 RNA-interacting proteins reveals that the DEAD-box RNA helicase DDX6 binds the DB1 and DB2 3′ UTR structures 
RNA Biology  2011;8(6):1173-1186.
Dengue virus (DENV) is a rapidly re-emerging flavivirus that causes dengue fever (DF), dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), diseases for which there are no available therapies or vaccines. The DENV-2 positive-strand RNA genome contains 5′ and 3′ untranslated regions (UTRs) that have been shown to form secondary structures required for virus replication and interaction with host cell proteins. In order to comprehensively identify host cell factors that bind the DENV-2 UTRs, we performed RNA chromatography, using the DENV-2 5′ and 3′ UTRs as “bait”, combined with quantitative mass spectrometry. We identified several proteins, including DDX6, G3BP1, G3BP2, Caprin1 and USP10, implicated in P body (PB) and stress granule (SG) function, and not previously known to bind DENV RNAs. Indirect immunofluorescence microscopy showed these proteins to colocalize with the DENV replication complex. Moreover, DDX6 knockdown resulted in reduced amounts of infectious particles and viral RNA in tissue culture supernatants following DENV infection. DDX6 interacted with DENV RNA in vivo during infection and in vitro this interaction was mediated by the DB1 and DB2 structures in the 3′ UTR, possibly by formation of a pseudoknot structure. Additional experiments demonstrate that, in contrast to DDX6, the SG proteins G3BP1, G3BP2, Caprin1 and USP10 bind to the variable region (VR) in the 3′ UTR. These results suggest that the DENV-2 3′ UTR is a site for assembly of PB and SG proteins and, for DDX6, assembly on the 3′ UTR is required for DENV replication.
PMCID: PMC3256426  PMID: 21957497
DDX6; dengue; host factors; RNA; stress granules
16.  The Kinase Inhibitor SFV785 Dislocates Dengue Virus Envelope Protein from the Replication Complex and Blocks Virus Assembly 
PLoS ONE  2011;6(8):e23246.
Dengue virus (DENV) is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785), which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.
PMCID: PMC3157368  PMID: 21858043
17.  Factors affecting reproducibility between genome-scale siRNA-based screens 
Journal of biomolecular screening  2010;15(7):735-747.
RNA interference-based screening is a powerful new genomic technology which addresses gene function en masse. To evaluate factors influencing hit list composition and reproducibility, we performed two identically designed small interfering RNA (siRNA)-based, whole genome screens for host factors supporting yellow fever virus infection. These screens represent two separate experiments completed five months apart and allow the direct assessment of the reproducibility of a given siRNA technology when performed in the same environment. Candidate hit lists generated by sum rank, median absolute deviation, z-score, and strictly standardized mean difference were compared within and between whole genome screens. Application of these analysis methodologies within a single screening dataset using a fixed threshold equivalent to a p-value ≤ 0.001 resulted in hit lists ranging from 82 to 1,140 members and highlighted the tremendous impact analysis methodology has on hit list composition. Intra- and inter-screen reproducibility was significantly influenced by the analysis methodology and ranged from 32% to 99%. This study also highlighted the power of testing at least two independent siRNAs for each gene product in primary screens. To facilitate validation we conclude by suggesting methods to reduce false discovery at the primary screening stage.
In this study we present the first comprehensive comparison of multiple analysis strategies, and demonstrate the impact of the analysis methodology on the composition of the “hit list”. Therefore, we propose that the entire dataset derived from functional genome-scale screens, especially if publicly funded, should be made available as is done with data derived from gene expression and genome-wide association studies.
PMCID: PMC3149892  PMID: 20625183
RNA interference; analysis; RNAi screen analysis; siRNA; RNAi; siRNA screening; sum rank; median absolute deviation; strictly standardized mean difference; genome-wide; whole-genome; comparison; overlap; hit list
19.  Alternative splicing in multiple sclerosis and other autoimmune diseases 
RNA Biology  2010;7(4):462-473.
Alternative splicing is a general mechanism for regulating gene expression that affects the RNA products of more than 90% of human genes. Not surprisingly, alternative splicing is observed among gene products of metazoan immune systems, which have evolved to efficiently recognize pathogens and discriminate between “self” and “non-self”, and thus need to be both diverse and flexible. In this review we focus on the specific interface between alternative splicing and autoimmune diseases, which result from a malfunctioning of the immune system and are characterized by the inappropriate reaction to self-antigens. Despite the widespread recognition of alternative splicing as one of the major regulators of gene expression, the connections between alternative splicing and autoimmunity have not been apparent. We summarize recent findings connecting splicing and autoimmune disease, and attempt to find common patterns of splicing regulation that may advance our understanding of autoimmune diseases and open new avenues for therapy.
PMCID: PMC3070910  PMID: 20639696
alternative splicing; autoimmune disease; SNP; multiple sclerosis; interleukin 7 receptor alpha chain
20.  SplicerAV: a tool for mining microarray expression data for changes in RNA processing 
BMC Bioinformatics  2010;11:108.
Over the past two decades more than fifty thousand unique clinical and biological samples have been assayed using the Affymetrix HG-U133 and HG-U95 GeneChip microarray platforms. This substantial repository has been used extensively to characterize changes in gene expression between biological samples, but has not been previously mined en masse for changes in mRNA processing. We explored the possibility of using HG-U133 microarray data to identify changes in alternative mRNA processing in several available archival datasets.
Data from these and other gene expression microarrays can now be mined for changes in transcript isoform abundance using a program described here, SplicerAV. Using in vivo and in vitro breast cancer microarray datasets, SplicerAV was able to perform both gene and isoform specific expression profiling within the same microarray dataset. Our reanalysis of Affymetrix U133 plus 2.0 data generated by in vitro over-expression of HRAS, E2F3, beta-catenin (CTNNB1), SRC, and MYC identified several hundred oncogene-induced mRNA isoform changes, one of which recognized a previously unknown mechanism of EGFR family activation. Using clinical data, SplicerAV predicted 241 isoform changes between low and high grade breast tumors; with changes enriched among genes coding for guanyl-nucleotide exchange factors, metalloprotease inhibitors, and mRNA processing factors. Isoform changes in 15 genes were associated with aggressive cancer across the three breast cancer datasets.
Using SplicerAV, we identified several hundred previously uncharacterized isoform changes induced by in vitro oncogene over-expression and revealed a previously unknown mechanism of EGFR activation in human mammary epithelial cells. We analyzed Affymetrix GeneChip data from over 400 human breast tumors in three independent studies, making this the largest clinical dataset analyzed for en masse changes in alternative mRNA processing. The capacity to detect RNA isoform changes in archival microarray data using SplicerAV allowed us to carry out the first analysis of isoform specific mRNA changes directly associated with cancer survival.
PMCID: PMC2838864  PMID: 20184770
21.  Correction of tau mis-splicing caused by FTDP-17 MAPT mutations by spliceosome-mediated RNA trans-splicing 
Human Molecular Genetics  2009;18(17):3266-3273.
Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the MAPT gene, encoding the tau protein that accumulates in intraneuronal lesions in a number of neurodegenerative diseases. Several FTDP-17 mutations affect alternative splicing and result in excess exon 10 (E10) inclusion in tau mRNA. RNA reprogramming using spliceosome-mediated RNA trans-splicing (SMaRT) could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and a pre-trans-splicing RNA molecule (PTM). However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. Excess E10 inclusion in FTDP-17 can be caused by intronic mutations destabilizing a stem-loop protecting the 5′ splice site at the E10/intron 10 junction. COS cells transfected with a minigene containing the intronic +14 mutation produce exclusively E10+ RNA. Generation of E10− RNA was restored after co-transfection with a PTM designed to exclude E10. Similar results were obtained with a target containing the exonic N279K mutation which strengthens a splicing enhancer within E10. Conversely, increase or decrease in E10 content was achieved by trans-splicing from a target carrying the Δ280K mutation, which weakens the same splicing enhancer. Thus E10 inclusion can be modulated by trans-splicing irrespective of the strength of the cis-splicing elements affected by FTDP-17 mutations. In conclusion, RNA trans-splicing could provide the basis of therapeutic strategies for impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements.
PMCID: PMC2722988  PMID: 19498037
22.  Tat-SF1 Is Not Required for Tat Transactivation but Does Regulate the Relative Levels of Unspliced and Spliced HIV-1 RNAs 
PLoS ONE  2009;4(5):e5710.
HIV-1 relies on several host proteins for productive viral transcription. HIV-1 Tat-specific factor 1 (Tat-SF1) is among these cofactors that were identified by in vitro reconstituted transcription reactions with immunodepleted nuclear extracts. At the onset of this work, the prevailing hypothesis was that Tat-SF1 was a required cofactor for the viral regulatory protein, Tat; however, this had not previously been formally tested in vivo.
Methodology/Principal Findings
To directly address the involvement of Tat-SF1 in HIV-1 gene expression, we depleted Tat-SF1 in HeLa cells by conventional expression of shRNAs and in T- Rex -293 cells containing tetracycline-inducible shRNAs targeting Tat-SF1. We achieved efficient depletion of Tat-SF1 and demonstrated that this did not affect cell viability. HIV-1 infectivity decreased in Tat-SF1-depleted cells, but only when multiple rounds of infection occurred. Neither Tat-dependent nor basal transcription from the HIV-1 LTR was affected by Tat-SF1 depletion, suggesting that the decrease in infectivity was due to a deficiency at a later step in the viral lifecycle. Finally, Tat-SF1 depletion resulted in an increase in the ratio of unspliced to spliced viral transcripts.
Tat-SF1 is not required for regulating HIV-1 transcription, but is required for maintaining the ratios of different classes of HIV-1 transcripts. These new findings highlight a novel, post-transcriptional role for Tat-SF1 in the HIV-1 life cycle.
PMCID: PMC2682658  PMID: 19479034
23.  hnRNP H and hnRNP F Complex with Fox2 To Silence Fibroblast Growth Factor Receptor 2 Exon IIIc▿ †  
Molecular and Cellular Biology  2008;28(17):5403-5419.
The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.
PMCID: PMC2519734  PMID: 18573884
24.  Dunning rat prostate adenocarcinomas and alternative splicing reporters: powerful tools to study epithelial plasticity in prostate tumors in vivo 
Using alternative splicing reporters we have previously observed mesenchymal epithelial transitions in Dunning AT3 rat prostate tumors. We demonstrate here that the Dunning DT and AT3 cells, which express epithelial and mesenchymal markers, respectively, represent an excellent model to study epithelial transitions since these cells recapitulate gene expression profiles observed during human prostate cancer progression. In this manuscript we also present the development of two new tools to study the epithelial transitions by imaging alternative splicing decisions: a bichromatic fluorescence reporter to evaluate epithelial transitions in culture and in vivo, and a luciferase reporter to visualize the distribution of mesenchymal epithelial transitions in vivo.
Electronic supplementary material
The online version of this article (doi:10.1007/s10585-008-9186-y) contains supplementary material, which is available to authorized users.
PMCID: PMC2471395  PMID: 18523850
DT; AT3; Dunning; FGFR2; Alternative splicing; EMT; MET; Imaging
25.  Initiation of Human Immunodeficiency Virus Type 1 (HIV-1) Transcription is Inhibited by Noncytolytic CD8+ Suppression 
The replication of human immunodeficiency virus type 1 (HIV-1) can be inhibited by noncytolytic CD8+ T cell mediated suppression, an immune response that specifically targets HIV-1 gene expression. Clinical studies demonstrate that this immune response may play an important role in the host defense against HIV infection. In this study, we examined the distinct steps in viral gene expression for inhibition by noncytolytic CD8+ T cells. A primary HIV-1 infection system of CD4+ enriched peripheral blood mononuclear cells was utilized to examine the HIV-1 life cycle as a relevant ex vivo system. Established CD8+ T cell lines from two HIV+ long-term nonprogressors were used to examine differences at the level of transcriptional initiation and elongation of the HIV genome. This infection system coupled with the results from real-time measurement of newly transcribed RNA transcripts determined that there was a significant decrease (5-8 fold) in short intracellular viral RNA transcripts. These data strongly favor a role for the initiation of virus transcription in noncytolytic CD8+ T cell mediated suppression.
PMCID: PMC2675544  PMID: 19440453

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