Interferon-Inducible MicroRNA miR-128 Modulates HIV-1 Replication by Targeting TNPO3 mRNA.

The HIV/AIDS pandemic remains an important threat to human health. We have recently demonstrated that a novel microRNA (miR), miR-128, represses retrotransposon long interspaced element 1 (L1) by a dual mechanism, namely, by directly targeting the coding region of the L1 RNA and by repressing a required nuclear import factor (TNPO1). We have further determined that miR-128 represses the expression of all three TNPO proteins (transportins TNPO1, TNPO2, and TNPO3). Here, we establish that miR-128 also influences HIV-1 replication by repressing TNPO3, a factor that regulates HIV-1 nuclear import and viral; replication of TNPO3 is well established to regulate HIV-1 nuclear import and viral replication. Here, we report that type I interferon (IFN)-inducible miR-128 directly targets two sites in the TNPO3 mRNA, significantly downregulating TNPO3 mRNA and protein expression levels. Challenging miR-modulated Jurkat cells or primary CD4+ T-cells with wild-type (WT), replication-competent HIV-1 demonstrated that miR-128 reduces viral replication and delays spreading of infection. Manipulation of miR-128 levels in HIV-1 target cell lines and in primary CD4+ T-cells by overexpression or knockdown showed that reduction of TNPO3 levels by miR-128 significantly affects HIV-1 replication but not murine leukemia virus (MLV) infection and that miR-128 modulation of HIV-1 replication is reduced with TNPO3-independent HIV-1 virus, suggesting that miR-128-indued TNPO3 repression contributes to the inhibition of HIV-1 replication. Finally, we determine that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Thus, we have established a novel role of miR-128 in antiviral defense in human cells, namely inhibiting HIV-1 replication by altering the cellular milieu through targeting factors that include TNPO3.IMPORTANCE HIV-1 is the causative agent of AIDS. During HIV-1 infection, type I interferons (IFNs) are induced, and their effectors limit HIV-1 replication at multiple steps in its life cycle. However, the cellular targets of INFs are still largely unknown. In this study, we identified the interferon-inducible microRNA (miR) miR-128, a novel antiviral mediator that suppresses the expression of the host gene TNPO3, which is known to modulate HIV-1 replication. Notably, we observe that anti-miR-128 partly neutralizes the IFN-mediated block of HIV-1. Elucidation of the mechanisms through which miR-128 impairs HIV-1 replication may provide novel candidates for the development of therapeutic interventions.

miR-128 Restriction of LINE-1 (L1) Retrotransposition Is Dependent on Targeting hnRNPA1 mRNA.

The majority of the human genome is made of transposable elements, giving rise to interspaced repeats, including Long INterspersed Element-1s (LINE-1s or L1s). L1s are active human transposable elements involved in genomic diversity and evolution; however, they can also contribute to genomic instability and diseases. L1s require host factors to complete their life cycles, whereas the host has evolved numerous mechanisms to restrict L1-induced mutagenesis. Restriction mechanisms in somatic cells include methylation of the L1 promoter, anti-viral factors and RNA-mediated processes such as small RNAs. microRNAs (miRNAs or miRs) are small non-coding RNAs that post-transcriptionally repress multiple target genes often found in the same cellular pathways. We have recently established that miR-128 functions as a novel restriction factor inhibiting L1 mobilization in somatic cells. We have further demonstrated that miR-128 functions through a dual mechanism; by directly targeting L1 RNA for degradation and indirectly by inhibiting a cellular co-factor which L1 is dependent on to transpose to new genomic locations (TNPO1). Here, we add another piece to the puzzle of the enigmatic L1 lifecycle. We show that miR-128 also inhibits another key cellular factor, hnRNPA1 (heterogeneous nuclear ribonucleoprotein A1), by significantly reducing mRNA and protein levels through direct interaction with the coding sequence (CDS) of hnRNPA1 mRNA. In addition, we demonstrate that repression of hnRNPA1 using hnRNPA1-shRNA significantly decreases de novo L1 retro-transposition and that induced hnRNPA1 expression enhances L1 mobilization. Furthermore, we establish that hnRNPA1 is a functional target of miR-128. Finally, we determine that induced hnRNPA1 expression in miR-128-overexpressing cells can partly rescue the miR-128-induced repression of L1's ability to transpose to different genomic locations. Thus, we have identified an additional mechanism by which miR-128 represses L1 retro-transposition and mediates genomic stability.

miR-128 inhibits telomerase activity by targeting TERT mRNA.

Telomerase is a unique cellular reverse transcriptase (RT) essential for maintaining telomere stability and required for the unlimited proliferation of cancer cells. The limiting determinant of telomerase activity is the catalytic component TERT, and TERT expression is closely correlated with telomerase activity and cancer initiation and disease progression. For this reason the regulation of TERT levels in the cell is of great importance. microRNAs (miRs) function as an additional regulatory level in cells, crucial for defining expression boundaries, proper cell fate decisions, cell cycle control, genome integrity, cell death and metastasis. We performed an anti-miR library screen to identity novel miRs, which participate in the control of telomerase. We identified the tumor suppressor miR (miR-128) as a novel endogenous telomerase inhibitor and determined that miR-128 significantly reduces the mRNA and protein levels of Tert in a panel of cancer cell lines. We further evaluated the mechanism by which miR-128 regulates TERT and demonstrated that miR-128 interacts directly with the coding sequence of TERT mRNA in both HeLa cells and teratoma cells. Interestingly, the functional miR-128 binding site in TERT mRNA, is conserved between TERT and the other cellular reverse transcriptase encoded by Long Interspersed Elements-1 (LINE-1 or L1), which can also contribute to the oncogenic phenotype of cancer. This finding supports the novel idea that miRs may function in parallel pathways to inhibit tumorigenesis, by regulating a group of enzymes (such as RT) by targeting conserved binding sites in the coding region of both enzymes.

MicroRNA miR-128 represses LINE-1 (L1) retrotransposition by down-regulating the nuclear import factor TNPO1.

Repetitive elements, including LINE-1 (L1), comprise approximately half of the human genome. These elements can potentially destabilize the genome by initiating their own replication and reintegration into new sites (retrotransposition). In somatic cells, transcription of L1 elements is repressed by distinct molecular mechanisms, including DNA methylation and histone modifications, to repress transcription. Under conditions of hypomethylation (e.g. in tumor cells), a window of opportunity for L1 derepression arises, and additional restriction mechanisms become crucial. We recently demonstrated that the microRNA miR-128 represses L1 activity by directly binding to L1 ORF2 RNA. In this study, we tested whether miR-128 can also control L1 activity by repressing cellular proteins important for L1 retrotransposition. We found that miR-128 targets the 3' UTR of nuclear import factor transportin 1 (TNPO1) mRNA. Manipulation of miR-128 and TNPO1 levels demonstrated that induction or depletion of TNPO1 affects L1 retrotransposition and nuclear import of an L1-ribonucleoprotein complex (using L1-encoded ORF1p as a proxy for L1-ribonucleoprotein complexes). Moreover, TNPO1 overexpression partially reversed the repressive effect of miR-128 on L1 retrotransposition. Our study represents the first description of a protein factor involved in nuclear import of the L1 element and demonstrates that miR-128 controls L1 activity in somatic cells through two independent mechanisms: direct binding to L1 RNA and regulation of a cellular factor necessary for L1 nuclear import and retrotransposition.

Quantification of Oligonucleotide Association with miRNA-Argonaute Complexes In Vitro.

A major challenge in the development of oligonucleotide-based microRNA (miRNA) inhibitors for therapeutic applications is the identification of candidate designs with strong affinity for the target miRNA in the context of the Argonaute complex. To this effect, distinct chemical modifications are employed along the length of the oligonucleotide aimed at strengthening the interactions with the target miRNA. However, the modification chemistry and placement can inadvertently affect the intrinsic ability of the oligonucleotide to pair with its target in the context of Argonaute. To facilitate the design of potent oligonucleotides, we developed a sensitive high-throughput methodology to compare anti-miR compounds for their ability to associate with the miRNA/Argonaute complex.

Determination of Anti-miR Association with miRNA/Argonaute Complexes In Vivo.

Aberrant expression of microRNAs (miRNAs) has been causatively linked to multiple disease pathologies while pharmacological inhibition of overexpressed miRNAs by modified oligonucleotides, termed anti-miRs, has been shown to ameliorate the disease phenotype. Anti-miRs are also widely used in academia to define miRNA-mediated regulation of gene networks in vitro and in vivo. Here, we describe a methodology that allows the determination of the physical association of miRNA inhibitors and their targets in the context of the Argonaute complex in vivo, providing unprecedented insight into the physiological interactions of anti-miRs and the miRNA machinery.

Transposable elements and miRNA: Regulation of genomic stability and plasticity.

Transposable elements, the class of mobile DNA sequences that change their copies or positions within the genome have an ever increasing role in shaping the genetic and evolutionary landscape. Approximately half of the mammalian genome is composed of repetitive elements, including LINE-1 (L1) elements. Because of their ability to "copy and paste" into other regions of the genome, their activation represent an opportunity as well as a threat, as L1-induced mutations results in genomic instability and plasticity. On one hand L1 retrotransposition and integration fosters genomic diversity and on the other, de-repressed L1 functions as a driver of diseases such as cancer. The regulation of L1 is an area of intense research and novel epigenetic mechanisms have recently been discovered to now include DNA methylation, histone modifications, and miR-induced L1 silencing. During development, reprogramming and in transformed cells, specific classes of repetitive elements are upregulated, presumably due to the loss of epigenetic regulation in this process, increasing the risk of L1-induced mutations. Here we discuss how miR regulation of L1 activation fits into the complex picture of L1 repression in somatic cells and touch on some of the possible implications.

miR-128 represses L1 retrotransposition by binding directly to L1 RNA.

Long interspersed element 1 (LINE-1 or L1) retrotransposons compose 17% of the human genome. Active L1 elements are capable of replicative transposition (mobilization) and can act as drivers of genetic diversity. However, this mobilization is mutagenic and may be detrimental to the host, and therefore it is under strict control. Somatic cells usually silence L1 activity by DNA methylation of the L1 promoter. In hypomethylated cells, such as cancer cells and induced pluripotent stem cells (iPSCs), a window of opportunity for L1 reactivation emerges, and with it comes an increased risk of genomic instability and tumorigenesis. Here we show that miR-128 represses new retrotransposition events in human cancer cells and iPSCs by binding directly to L1 RNA. Thus, we have identified and characterized a new function of microRNAs: mediating genomic stability by suppressing the mobility of endogenous retrotransposons.

Anti-miRs competitively inhibit microRNAs in Argonaute complexes.

MicroRNAs (miRNAs), small RNA molecules that post-transcriptionally regulate mRNA expression, are crucial in diverse developmental and physiological programs and their misregulation can lead to disease. Chemically modified oligonucleotides have been developed to modulate miRNA activity for therapeutic intervention in disease settings, but their mechanism of action has not been fully elucidated. Here we show that the miRNA inhibitors (anti-miRs) physically associate with Argonaute proteins in the context of the cognate target miRNA in vitro and in vivo. The association is mediated by the seed region of the miRNA and is sensitive to the placement of chemical modifications. Furthermore, the targeted miRNAs are stable and continue to be associated with Argonaute. Our results suggest that anti-miRs specifically associate with Argonaute-bound miRNAs, preventing association with target mRNAs, which leads to subsequent stabilization and thus increased expression of the targeted mRNAs.

Functional genomic analysis of the let-7 regulatory network in Caenorhabditis elegans.

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7-dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.

Autoregulation of microRNA biogenesis by let-7 and Argonaute.

MicroRNAs (miRNAs) comprise a large family of small RNA molecules that post-transcriptionally regulate gene expression in many biological pathways. Most miRNAs are derived from long primary transcripts that undergo processing by Drosha to produce ~65-nucleotide precursors that are then cleaved by Dicer, resulting in the mature 22-nucleotide forms. Serving as guides in Argonaute protein complexes, mature miRNAs use imperfect base pairing to recognize sequences in messenger RNA transcripts, leading to translational repression and destabilization of the target messenger RNAs. Here we show that the miRNA complex also targets and regulates non-coding RNAs that serve as substrates for the miRNA-processing pathway. We found that the Argonaute protein in Caenorhabditis elegans, ALG-1, binds to a specific site at the 3' end of let-7 miRNA primary transcripts and promotes downstream processing events. This interaction is mediated by mature let-7 miRNA through a conserved complementary site in its own primary transcript, thus creating a positive-feedback loop. We further show that ALG-1 associates with let-7 primary transcripts in nuclear fractions. Argonaute also binds let-7 primary transcripts in human cells, demonstrating that the miRNA pathway targets non-coding RNAs in addition to protein-coding messenger RNAs across species. Moreover, our studies in C. elegans reveal a novel role for Argonaute in promoting biogenesis of a targeted transcript, expanding the functions of the miRNA pathway in gene regulation. This discovery of autoregulation of let-7 biogenesis establishes a new mechanism for controlling miRNA expression.

Comprehensive identification of miRNA target sites in live animals.

MicroRNAs (miRNAs) are small RNA molecules that posttranscriptionally regulate the expression of protein-coding genes. The mature miRNAs are loaded into Argonaute-containing protein complexes (miRISC, miRNA Induced S ilencing Complex), and guide these complexes to the 3' UTR of targeted mRNA transcripts via base-pairing interactions. However, the imperfect complementarity that characterizes the interactions between animal miRNAs and target sites complicates the identification of direct target genes. We developed a biochemical method to identify on a large scale the target sequences recognized by miRISC in vivo. The mRNA sites bound by miRISC are stabilized by cross-linking and isolated by immunoprecipitation of Argonaute-containing complexes. The bound RNA molecules are trimmed to the regions protected by Argonaute, subjected to a series of isolation and linker ligation steps and identified by high-throughput sequencing methods.

Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans.

MicroRNAs (miRNAs) regulate gene expression by guiding Argonaute proteins to specific target mRNA sequences. Identification of bona fide miRNA target sites in animals is challenging because of uncertainties regarding the base-pairing requirements between miRNA and target as well as the location of functional binding sites within mRNAs. Here we present the results of a comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the Argonaute protein ALG-1 in C. elegans. Using cross-linking and ALG-1 immunoprecipitation coupled with high-throughput sequencing (CLIP-seq), we identified extensive ALG-1 interactions with specific 3' untranslated region (UTR) and coding exon sequences and discovered features that distinguish miRNA complex binding sites in 3' UTRs from those in other genic regions. Furthermore, our analyses revealed a striking enrichment of Argonaute binding sites in genes important for miRNA function, suggesting an autoregulatory role that may confer robustness to the miRNA pathway.

Stat3 and Stat4 direct development of IL-17-secreting Th cells.

IL-17-secreting CD4(+) T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFbeta1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFbeta1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORgammat expression in TGFbeta1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFbeta1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types.

T-bet is a critical determinant in the instability of the IL-17-secreting T-helper phenotype.

IL-23, an IL-12-related cytokine, induces an IL-17-secreting T-helper phenotype that is involved in autoimmune diseases and host defense against certain pathogens. Although the transcription factors required for development of IL-23-stimulated cells are unknown, we show that T-bet is a critical negative regulator of the IL-23-primed T-cell phenotype, which we term Th1beta. Th1 or Th1beta Tbx21-/- cultures secrete higher than WT levels of IL-17 in response to T-cell receptor (TCR) or IL-23 + IL-18 stimulation. Ectopic T-bet expression in Th1beta cells promotes IFN-gamma secretion but decreases IL-17 production. Although antigen-receptor stimulation of Th1beta cells stimulates IL-17 production, it also induces the IFN-gamma-independent expression of T-bet and progression to a Th1 cytokine secretion pattern. T-bet is required for the progression to the Th1 phenotype, because Tbx21-/- Th1beta cultures maintain the IL-17-secreting phenotype after 2 weeks of culture. Addition of IFN-gamma to Tbx21-/- Th1beta cultures cannot recover the progression to the Th1 phenotype, suggesting T-bet, rather than IFN-gamma, mediates Th1beta to Th1 progression. The transient nature of the Th1beta phenotype suggests that these cells are a component of type I immunity and that T-bet expression is a critical determinant of Th1 versus Th1beta cell fate.

A crucial role for T-bet in selectin ligand expression in T helper 1 (Th1) cells.

Proinflammatory T helper 1 (Th1) cells express high levels of carbohydrate ligands for the endothelial selectins, but the molecular basis for this phenotype is incompletely understood. We document here a significant role in selectin ligand formation for the recently described Th1 transcription factor T-bet. Th1 cells generated from T-bet-/- mice showed significantly lower levels of ligands for both E-selectin and P-selectin, compared with wild-type (WT) Th1 cells. Enforced expression of T-bet in WT Th0 cells only modestly up-regulated P-selectin ligands and had no effect on E-selectin ligands. To define a mechanism for the defects observed in T-bet-/- mice, we examined expression of glycosyltransferases involved in selectin ligand biosynthesis. T-bet-/- Th1 cells expressed significantly lower levels of core 2 beta1,6 N-acetylglucosaminyltransferase I (C2GlcNAcT-I), but no differences in levels of alpha 2,3-sialyltransferase IV (ST3Gal-IV). Further, we show that T-bet is responsible for the signal transducer and activator of transcription 4 (Stat4)-independent increase in Th1 cells of fucosyltransferase VII (FucT-VII). We also identify ST3Gal-VI, which is thought to play an important role in E- and P-selectin ligand formation, as an interleukin 12 (IL-12)-regulated, T-bet-dependent gene. These data show that T-bet controls selectin ligand formation in Th1 cells via control of expression of multiple key enzymes in response to IL-12 signaling and establishes an independent transcriptional pathway for control of Th1 cell traffic.

H-Ras and phosphoinositide 3-kinase cooperate to induce alpha(1,3)-fucosyltransferase VII expression in Jurkat T cells.

The alpha(1,3)-fucosyltransferase FucT-VII is essential for the biosynthesis of selectin ligands, but the signaling pathways mediating FucT-VII induction in T cells and other lymphocytes are poorly understood. We have shown previously that sustained activation of Ras in Jurkat T cells induces FucT-VII transcription, which requires the Raf-MEK-ERK pathway. In this study we report that FucT-VII induction is specific to the H-Ras isoform. Jurkat T cells retrovirally transduced with constitutively active H-Ras but not N- or K-Ras up-regulated expression of FucT-VII. Pharmacological inhibition studies also revealed that phosphoinositide 3-kinase (PI3K) activity is required for H-Ras-mediated FucT-VII induction. However, the ability of H-Ras to selectively induce FucT-VII is not a function of the inability of the N- or K-Ras isoforms to activate Raf or PI3K pathways. The use of effector-loop domain mutants of H-Ras, which are impaired for their ability to interact selectively with individual effectors alone or in combination with active Raf, indicated that induction of FucT-VII requires the concomitant activation of at least three signaling pathways. These studies show that H-Ras mediates FucT-VII induction in Jurkat T cells via the activation of the Raf, PI3K, and a distinct, H-Ras-specific effector signaling pathway.

Induction of FucT-VII by the Ras/MAP kinase cascade in Jurkat T cells.

Induction of the alpha1,3-fucosyltransferase FucT-VII in T lymphocytes is crucial for selectin ligand formation, but the signaling and transcriptional pathways that govern FucT-VII expression are unknown. Here, using a novel, highly phorbol myristate acetate (PMA)-responsive variant of the Jurkat T-cell line, we identify Ras and downstream mitogen-activated protein (MAP) kinase pathways as essential mediators of FucT-VII gene expression. PMA induced FucT-VII in only a subset of treated cells, similar to expression of FucT-VII in normal activated CD4 T cells. Introduction of constitutively active Ras or Raf by recombinant retroviruses induced FucT-VII expression only in that subset of cells expressing the highest levels of Ras, suggesting that induction of FucT-VII required a critical threshhold of Ras signaling. Both PMA treatment and introduction of active Ras led to rolling on E-selectin. Pharmacologic inhibition studies confirmed the involvement of the classic Ras-Raf-MEK-extracellular signal-regulated kinase (Ras-Raf-MEK-ERK) pathway in FucT-VII induction by PMA, Ras, and Raf. These studies also revealed a second, Ras-induced, Raf-1-independent pathway that participated in induction of FucT-VII. Strong activation of Ras represents a major pathway for induction of FucT-VII gene expression in T cells.