RESUMEN
The NRF2 transcription factor controls a cell stress program that is implicated in cancer and there is great interest in targeting NRF2 for therapy. We show that NRF2 activity depends on Fructosamine-3-kinase (FN3K)-a kinase that triggers protein de-glycation. In its absence, NRF2 is extensively glycated, unstable, and defective at binding to small MAF proteins and transcriptional activation. Moreover, the development of hepatocellular carcinoma triggered by MYC and Keap1 inactivation depends on FN3K in vivo. N-acetyl cysteine treatment partially rescues the effects of FN3K loss on NRF2 driven tumor phenotypes indicating a key role for NRF2-mediated redox balance. Mass spectrometry reveals that other proteins undergo FN3K-sensitive glycation, including translation factors, heat shock proteins, and histones. How glycation affects their functions remains to be defined. In summary, our study reveals a surprising role for the glycation of cellular proteins and implicates FN3K as targetable modulator of NRF2 activity in cancer.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Carcinoma Hepatocelular/patología , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Glicosilación , Células HEK293 , Células Hep G2 , Xenoinjertos , Humanos , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transducción GenéticaRESUMEN
The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo signaling pathway activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivation. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.
Asunto(s)
Vía de Señalización Hippo , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Animales , Humanos , Ratones , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Paclitaxel/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al GTP rap/metabolismo , Proteínas de Unión al GTP rap/genética , Proteína de Unión al GTP rhoA/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , FosforilaciónRESUMEN
The activation of memory T cells is a very rapid and concerted cellular response that requires coordination between cellular processes in different compartments and on different time scales. In this study, we use ribosome profiling and deep RNA sequencing to define the acute mRNA translation changes in CD8 memory T cells following initial activation events. We find that initial translation enables subsequent events of human and mouse T cell activation and expansion. Briefly, early events in the activation of Ag-experienced CD8 T cells are insensitive to transcriptional blockade with actinomycin D, and instead depend on the translation of pre-existing mRNAs and are blocked by cycloheximide. Ribosome profiling identifies â¼92 mRNAs that are recruited into ribosomes following CD8 T cell stimulation. These mRNAs typically have structured GC and pyrimidine-rich 5' untranslated regions and they encode key regulators of T cell activation and proliferation such as Notch1, Ifngr1, Il2rb, and serine metabolism enzymes Psat1 and Shmt2 (serine hydroxymethyltransferase 2), as well as translation factors eEF1a1 (eukaryotic elongation factor α1) and eEF2 (eukaryotic elongation factor 2). The increased production of receptors of IL-2 and IFN-γ precedes the activation of gene expression and augments cellular signals and T cell activation. Taken together, we identify an early RNA translation program that acts in a feed-forward manner to enable the rapid and dramatic process of CD8 memory T cell expansion and activation.
Asunto(s)
Glicina Hidroximetiltransferasa , Interleucina-2 , Regiones no Traducidas 5' , Animales , Linfocitos T CD8-positivos , Cicloheximida/metabolismo , Dactinomicina/metabolismo , Glicina Hidroximetiltransferasa/genética , Glicina Hidroximetiltransferasa/metabolismo , Humanos , Memoria Inmunológica , Interleucina-2/metabolismo , Activación de Linfocitos , Células T de Memoria , Ratones , Factor 2 de Elongación Peptídica/genética , Factor 2 de Elongación Peptídica/metabolismo , Factores de Elongación de Péptidos/genética , Pirimidinas/metabolismo , ARN Mensajero/genética , Serina/genéticaRESUMEN
The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.
Asunto(s)
Regiones no Traducidas 5'/genética , Factor 4A Eucariótico de Iniciación/metabolismo , G-Cuádruplex , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Biosíntesis de Proteínas , Animales , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Secuencia de Bases , Línea Celular Tumoral , Epigénesis Genética , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Motivos de Nucleótidos , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Biosíntesis de Proteínas/efectos de los fármacos , Ribosomas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Triterpenos/farmacologíaRESUMEN
MicroRNAs (miRNAs) are known to have a role in gene regulation that is closely integrated into the pathways that control virtually all fundamental cell processes of growth, differentiation, metabolism, and death. Whether silencing RNAs and the cellular pathways that generate them are also used in antiviral defense in higher eukaryotes, as they are in plants and lower eukaryotes, has been the subject of much study. Results to date point to a complex interplay between viruses and vertebrate host cells that can vary considerably among different viruses. Here, we review current knowledge regarding interactions between HIV-1 and host cell RNA silencing mechanisms. Important questions in this field remain unresolved, including whether HIV-1 itself encodes small silencing RNAs that might either promote or repress its replication, whether host cell miRNAs can directly target viral transcripts or can alter the course of infection indirectly through effects on cellular genes necessary for viral replication, and whether HIV-1 produces proteins or RNAs that suppress the host-silencing pathway. We summarize evidence and controversies related to the potential role of RNA silencing pathways as a defense against HIV-1 infection.
Asunto(s)
Infecciones por VIH/genética , Infecciones por VIH/prevención & control , Interferencia de ARN/fisiología , Animales , Humanos , MicroARNs/genética , ARN Interferente PequeñoRESUMEN
The Hippo signaling is instrumental in regulating organ size, regeneration, and carcinogenesis. The cytoskeleton emerges as a primary Hippo signaling modulator. Its structural alterations in response to environmental and intrinsic stimuli control Hippo kinase cascade activity. However, the precise mechanisms underlying the cytoskeleton regulation of Hippo signaling are not fully understood. RAP2 GTPase is known to mediate the mechanoresponses of Hippo signaling via activating the core Hippo kinases LATS1/2 through MAP4Ks and MST1/2. Here we show the pivotal role of the reciprocal regulation between RAP2 GTPase and the cytoskeleton in Hippo signaling. RAP2 deletion undermines the responses of the Hippo pathway to external cues tied to RhoA GTPase inhibition and actin cytoskeleton remodeling, such as energy stress and serum deprivation. Notably, RhoA inhibitors and actin disruptors fail to activate LATS1/2 effectively in RAP2-deficient cells. RNA sequencing highlighted differential regulation of both actin and microtubule networks by RAP2 gene deletion. Consistently, Taxol, a microtubule-stabilizing agent, was less effective in activating LATS1/2 and inhibiting cell growth in RAP2 and MAP4K4/6/7 knockout cells. In summary, our findings position RAP2 as a central integrator of cytoskeletal signals for Hippo signaling, which offers new avenues for understanding Hippo regulation and therapeutic interventions in Hippo-impaired cancers.
RESUMEN
The TAR RNA binding protein, TRBP, is a cellular double-stranded RNA (dsRNA) binding protein that can promote the replication of HIV-1 through interactions with the viral TAR element as well as with cellular proteins that affect the efficiency of translation of viral transcripts. The structured TAR element, present on all viral transcripts, can impede efficient translation either by sterically blocking access of translation initiation factors to the 5'-cap or by activating the dsRNA-dependent kinase, PKR. Several mechanisms by which TRBP can facilitate translation of viral transcripts have been proposed, including the binding and unwinding of TAR and the suppression of PKR activation. Further, TRBP has been identified as a cofactor of Dicer in the processing of microRNAs (miRNAs), and sequestration of TRBP by TAR in infected cells has been proposed as a viral countermeasure to potential host cell RNA interference-based antiviral activities. Here, we have addressed the relative importance of these various roles for TRBP in HIV-1 replication. Using Jurkat T cells, primary human CD4(+) T cells, and additional cultured cell lines, we show that depletion of TRBP has no effect on viral replication when PKR activation is otherwise blocked. Moreover, the presence of TAR-containing mRNAs does not affect the efficacy of cellular miRNA silencing pathways. These results establish that TRBP, when expressed at physiological levels, promotes HIV-1 replication mainly by suppressing the PKR-mediated antiviral response, while its contribution to HIV-1 replication through PKR-independent pathways is minimal.
Asunto(s)
Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/patogenicidad , Proteínas de Unión al ARN/metabolismo , Replicación Viral , eIF-2 Quinasa/antagonistas & inhibidores , Western Blotting , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/virología , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , Infecciones por VIH/genética , Células HeLa , Humanos , Células Jurkat , MicroARNs/fisiología , Fosforilación , Unión Proteica , ARN Bicatenario/metabolismo , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasa III/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
The eIF4E translation initiation factor has oncogenic properties and concordantly, the inhibitory eIF4E-binding protein (4EBP1) is considered a tumor suppressor. The exact molecular effects of 4EBP1 activation in cancer are still unknown. Surprisingly, 4EBP1 is a target of genomic copy number gains (Chr. 8p11) in breast and lung cancer. We noticed that 4EBP1 gains are genetically linked to gains in neighboring genes, including WHSC1L1 and FGFR1. Our results show that FGFR1 gains act to attenuate the function of 4EBP1 via PI3K-mediated phosphorylation at Thr37/46, Ser65, and Thr70 sites. This implies that not 4EBP1 but instead FGFR1 is the genetic target of Chr. 8p11 gains in breast and lung cancer. Accordingly, these tumors show increased sensitivity to FGFR1 and PI3K inhibition, and this is a therapeutic vulnerability through restoring the tumor-suppressive function of 4EBP1. Ribosome profiling reveals genes involved in insulin signaling, glucose metabolism, and the inositol pathway to be the relevant translational targets of 4EBP1. These mRNAs are among the top 200 translation targets and are highly enriched for structure and sequence motifs in their 5'UTR, which depends on the 4EBP1-EIF4E activity. In summary, we identified the translational targets of 4EBP1-EIF4E that facilitate the tumor suppressor function of 4EBP1 in cancer.
RESUMEN
Inhibition of the eIF4A RNA helicase with silvestrol and related compounds is emerging as a powerful anti-cancer strategy. We find that a synthetic silvestrol analogue (CR-1-31 B) has nanomolar activity across many cancer cell lines. It is especially active against aggressive MYC+/BCL2+ B cell lymphomas and this likely reflects the eIF4A-dependent translation of both MYC and BCL2. We performed a genome-wide CRISPR/Cas9 screen and identified mechanisms of resistance to this new class of therapeutics. We identify three negative NRF2 regulators (KEAP1, CUL3, CAND1) whose inactivation is sufficient to cause CR1-31-B resistance. NRF2 is known to alter the oxidation state of translation factors and cause a broad increase in protein production. We find that NRF2 activation particularly increases the translation of some eIF4A-dependent mRNAs and restores MYC and BCL2 production. We know that NRF2 functions depend on removal of sugar adducts by the frutosamine-3-kinase (FN3K). Accordingly, loss of FN3K results in NRF2 hyper-glycation and inactivation and resensitizes cancer cells to eIF4A inhibition. Together, our findings implicate NRF2 in the translation of eIF4A-dependent mRNAs and point to FN3K inhibition as a new strategy to block NRF2 functions in cancer.
RESUMEN
Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.
Asunto(s)
Adenocarcinoma/metabolismo , Factor 4A Eucariótico de Iniciación/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Regiones no Traducidas 5' , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenocarcinoma/tratamiento farmacológico , Animales , Línea Celular Tumoral , Cicloheximida/farmacología , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , G-Cuádruplex , Genes ras/genética , Humanos , Ratones , Ratones Desnudos , Mutación , Trasplante de Neoplasias , Oxidación-Reducción , Neoplasias Pancreáticas/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Proto-Oncogénicas c-met/genética , Proteínas Proto-Oncogénicas c-met/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Helicasas , Análisis de Secuencia de ARN , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcriptoma , Triterpenos/farmacología , Proteínas Señalizadoras YAP , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rac/metabolismo , Proteínas de Unión al GTP ral/genética , Proteínas de Unión al GTP ral/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
Follicular lymphoma (FL) is an incurable form of B cell lymphoma. Genomic studies have cataloged common genetic lesions in FL such as translocation t(14;18), frequent losses of chromosome 6q, and mutations in epigenetic regulators such as EZH2 Using a focused genetic screen, we identified SESTRIN1 as a relevant target of the 6q deletion and demonstrate tumor suppression by SESTRIN1 in vivo. Moreover, SESTRIN1 is a direct target of the lymphoma-specific EZH2 gain-of-function mutation (EZH2Y641X ). SESTRIN1 inactivation disrupts p53-mediated control of mammalian target of rapamycin complex 1 (mTORC1) and enables mRNA translation under genotoxic stress. SESTRIN1 loss represents an alternative to RRAGC mutations that maintain mTORC1 activity under nutrient starvation. The antitumor efficacy of pharmacological EZH2 inhibition depends on SESTRIN1, indicating that mTORC1 control is a critical function of EZH2 in lymphoma. Conversely, EZH2Y641X mutant lymphomas show increased sensitivity to RapaLink-1, a bifunctional mTOR inhibitor. Hence, SESTRIN1 contributes to the genetic and epigenetic control of mTORC1 in lymphoma and influences responses to targeted therapies.
Asunto(s)
Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Proteínas de Choque Térmico/genética , Linfoma Folicular/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Animales , Deleción Cromosómica , Cromosomas Humanos Par 6/genética , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Silenciador del Gen , Pruebas Genéticas , Genoma Humano , Proteínas de Choque Térmico/deficiencia , Humanos , Ratones , Mutación/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The posttranscriptional control of gene expression by microRNAs (miRNAs) is highly redundant, and compensatory effects limit the consequences of the inactivation of individual miRNAs. This implies that only a few miRNAs can function as effective tumor suppressors. It is also the basis of our strategy to define functionally relevant miRNA target genes that are not under redundant control by other miRNAs. We identified a functionally interconnected group of miRNAs that exhibited a reduced abundance in leukemia cells from patients with T cell acute lymphoblastic leukemia (T-ALL). To pinpoint relevant target genes, we applied a machine learning approach to eliminate genes that were subject to redundant miRNA-mediated control and to identify those genes that were exclusively targeted by tumor-suppressive miRNAs. This strategy revealed the convergence of a small group of tumor suppressor miRNAs on the Myb oncogene, as well as their effects on HBP1, which encodes a transcription factor. The expression of both genes was increased in T-ALL patient samples, and each gene promoted the progression of T-ALL in mice. Hence, our systematic analysis of tumor suppressor miRNA action identified a widespread mechanism of oncogene activation in T-ALL.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , Genes Supresores de Tumor , Genes myb/genética , MicroARNs/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Traslado Adoptivo , Animales , Inteligencia Artificial , Trasplante de Células Madre Hematopoyéticas , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Modelos Genéticos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/metabolismoRESUMEN
In many RNA silencing applications, there is a benefit to expressing multiple interfering RNAs simultaneously. This can be achieved by using a single RNA polymerase II promoter to express multiple micro(mi)RNA-formatted interfering RNAs that are arranged in a polycistronic cluster, mimicking the organization of naturally clustered, endogenous miRNAs. While RNA pol III promoters are often used to express individual short hairpin (sh) RNAs, we have recently shown that pol III promoters can also be used to drive polycistronic expression of miRNA-formatted interfering RNAs. Here, we present methods for the assembly of polycistronic miRNA expression vectors that use pol III promoters. In addition, we present methods for testing the potency and the level of expression of each of the individual miRNAs encoded in the construct.
Asunto(s)
MicroARNs/genética , Regiones Promotoras Genéticas , ARN Polimerasa III/genética , ARN Interferente Pequeño/genética , Secuencia de Bases , Clonación Molecular/métodos , Expresión Génica , Genes Reporteros , Luciferasas/biosíntesis , Luciferasas/genética , Datos de Secuencia Molecular , Familia de Multigenes , Interferencia de ARN , ARN Interferente Pequeño/biosíntesisRESUMEN
The therapeutic value of antiviral interfering RNAs could be improved by technologies that limit their expression to the infected cell population. The HIV-1 Tat-inducible viral LTR and LTR-containing chimeric promoters have previously been used to drive expression of antiviral RNAs and proteins directed against HIV-1. Here, we characterize an alternative promoter, consisting of a chicken ß-actin core promoter fused to the viral TAR element, for the conditional expression of interfering RNAs. This promoter, that we refer to as the CK-TAR promoter, can induce levels of silencing comparable to the viral LTR in response to Tat produced from co-transfected plasmids or during viral replication. While the CK-TAR promoter shows a modest level of basal activity, similar to the viral LTR, it is less responsive to the extracellular stimuli tested including LPS, TNFα, and PMA. The CK-TAR promoter is an alternative Tat-inducible promoter with the potential to minimize the risk of vector mobilization and to drive polycistronic expression of interfering RNAs.
Asunto(s)
Duplicado del Terminal Largo de VIH , Regiones Promotoras Genéticas , ARN Interferente Pequeño/biosíntesis , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Actinas/genética , Animales , Fusión Artificial Génica , Línea Celular , Pollos , HumanosRESUMEN
The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing.