NF90 modulates processing of a subset of human pri-miRNAs.

MicroRNAs (miRNAs) are predicted to regulate the expression of >60% of mammalian genes and play fundamental roles in most biological processes. Deregulation of miRNA expression is a hallmark of most cancers and further investigation of mechanisms controlling miRNA biogenesis is needed. The double stranded RNA-binding protein, NF90 has been shown to act as a competitor of Microprocessor for a limited number of primary miRNAs (pri-miRNAs). Here, we show that NF90 has a more widespread effect on pri-miRNA biogenesis than previously thought. Genome-wide approaches revealed that NF90 is associated with the stem region of 38 pri-miRNAs, in a manner that is largely exclusive of Microprocessor. Following loss of NF90, 22 NF90-bound pri-miRNAs showed increased abundance of mature miRNA products. NF90-targeted pri-miRNAs are highly stable, having a lower free energy and fewer mismatches compared to all pri-miRNAs. Mutations leading to less stable structures reduced NF90 binding while increasing pri-miRNA stability led to acquisition of NF90 association, as determined by RNA electrophoretic mobility shift assay (EMSA). NF90-bound and downregulated pri-miRNAs are embedded in introns of host genes and expression of several host genes is concomitantly reduced. These data suggest that NF90 controls the processing of a subset of highly stable, intronic miRNAs.

STAT3 induces G9a to exacerbate HER3 expression for the survival of epidermal growth factor receptor-tyrosine kinase inhibitors in lung cancers.

BACKGROUND: HER3 mediates drug resistance against epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), resulting in tumor relapse in lung cancers. Previously, we demonstrated that EGFR induces HER3 overexpression, which facilitates the formation of cancer stem-like tumorspheres. However, the cellular mechanism through which EGFR regulates HER3 expression remains unclear. We hypothesized that EGFR downstream of STAT3 participates in HER3 expression because STAT3 contributes to cancer stemness and survival of EGFR-TKI resistant cancers. METHODS: First, RNAseq was used to uncover potential genes involved in the formation of lung cancer HCC827-derived stem-like tumorspheres. EGFR-positive lung cancer cell lines, including HCC827, A549, and H1975, were individually treated with a panel containing 172 therapeutic agents targeting stem cell-associated genes to search for potential agents that could be applied against EGFR-positive lung cancers. In addition, gene knockdown and RNAseq were used to investigate molecular mechanisms through which STAT3 regulates tumor progression and the survival in lung cancer. RESULTS: BBI608, a STAT3 inhibitor, was a potential therapeutic agent that reduced the cell viability of EGFR-positive lung cancer cell lines. Notably, the inhibitory effects of BBI608 were similar with those associated with YM155, an ILF3 inhibitor. Both compounds reduced G9a-mediated HER3 expression. We also demonstrated that STAT3 upregulated G9a to silence miR-145-5p, which exacerbated HER3 expression in this study. CONCLUSIONS: The present study revealed that BBI608 could eradicate EGFR-positive lung cancers and demonstrated that STAT3 enhanced the expression of HER3 through miR-145-5p repression by G9a, indicating that STAT3 is a reliable therapeutic target against EGFR-TKI-resistant lung cancers.

RNA stability protein ILF3 mediates cytokine-induced angiogenesis.

Interleukin enhancer-binding factor 3 (ILF3), an RNA-binding protein, is best known for its role in innate immunity by participation in cellular antiviral responses. A role for ILF3 in angiogenesis is unreported. ILF3 expression in CD31+ capillaries of hypoxic cardiac tissue was detected by immunohistochemistry. Proangiogenic stimuli induce ILF3 mRNA and protein expression in cultured human coronary artery endothelial cells (hCAECs). Angiogenic indices, including proliferation, migration, and tube formation, are all significantly reduced in hCAECs when ILF3 is knocked down using small interfering RNA (siRNA), but are significantly increased when ILF3 is overexpressed using adenovirus. Protein and mRNA abundance of several angiogenic factors including CXCL1, VEGF, and IL-8 are decreased when ILF3 is knocked down by siRNA. These factors are increased when ILF3 is overexpressed by adenovirus. ILF3 is phosphorylated and translocates from the nucleus to the cytoplasm in response to angiogenic stimuli. Proangiogenic transcripts containing adenine and uridine-rich elements were bound to ILF3 through RNA immunoprecipitation. ILF3 stabilizes proangiogenic transcripts including VEGF, CXCL1, and IL-8 in hCAECs. Together these data suggest that in endothelial cells, the RNA stability protein, ILF3, plays a novel and central role in angiogenesis. Our working hypothesis is that ILF3 promotes angiogenesis through cytokine-inducible mRNA stabilization of proangiogenic transcripts.-Vrakas, C. N., Herman, A. B., Ray, M., Kelemen, S. E., Scalia, R., Autieri, M. V. RNA stability protein ILF3 mediates cytokine-induced angiogenesis.

EGFR-mediated interleukin enhancer-binding factor 3 contributes to formation and survival of cancer stem-like tumorspheres as a therapeutic target against EGFR-positive non-small cell lung cancer.

OBJECTIVES: YM155, an inhibitor of interleukin enhancer-binding factor 3 (ILF3), significantly suppresses cancer stemness property, implying that ILF3 contributes to cell survival of cancer stem cells. However, the molecular function of ILF3 inhibiting cancer stemness remains unclear. This study aimed to uncover the potential function of ILF3 involving in cell survival of epidermal growth factor receptor (EGFR)-positive lung stem-like cancer, and to investigate the potential role to improve the efficacy of anti-EGFR therapeutics. MATERIALS AND METHODS: The association of EGFR and ILF3 in expression and regulations was first investigated in this study. Lung cancer A549 cells with deprivation of ILF3 were created by the gene-knockdown method and then RNAseq was applied to identify the putative genes regulated by ILF3. Meanwhile, HCC827- and A549-derived cancer stem-like cells were used to investigate the role of ILF3 in the formation of cancer stem-like tumorspheres. RESULTS: We found that EGFR induced ILF3 expression, and YM155 reduced EGFR expression. The knockdown of ILF3 reduced not only EGFR expression in mRNA and protein levels, but also cell proliferation in vitro and in vivo, demonstrating that ILF3 may play an important role in contributing to cancer cell survival. Moreover, the knockdown and inhibition of ILF3 by shRNA and YM155, respectively, reduced the formation and survival of HCC827- and A549-derived tumorspheres through inhibiting ErbB3 (HER3) expression, and synergized the therapeutic efficacy of afatinib, a tyrosine kinase inhibitor, against EGFR-positive A549 lung cells. CONCLUSION: This study demonstrated that ILF3 plays an oncogenic like role in maintaining the EGFR-mediated cellular pathway, and can be a therapeutic target to improve the therapeutic efficacy of afatinib. Our results suggested that YM155, an ILF3 inhibitor, has the potential for utilization in cancer therapy against EGFR-positive lung cancers.

NF45 and NF90/NF110 coordinately regulate ESC pluripotency and differentiation.

While years of investigation have elucidated many aspects of embryonic stem cell (ESC) regulation, the contributions of post-transcriptional and translational mechanisms to the pluripotency network remain largely unexplored. In particular, little is known in ESCs about the function of RNA binding proteins (RBPs), the protein agents of post-transcriptional regulation. We performed an unbiased RNAi screen of RBPs in an ESC differentiation assay and identified two related genes, NF45 (Ilf2) and NF90/NF110 (Ilf3), whose knockdown promoted differentiation to an epiblast-like state. Characterization of NF45 KO, NF90 + NF110 KO, and NF110 KO ESCs showed that loss of NF45 or NF90 + NF110 impaired ESC proliferation and led to dysregulated differentiation down embryonic lineages. Additionally, we found that NF45 and NF90/NF110 physically interact and influence the expression of each other at different levels of regulation. Globally across the transcriptome, NF45 KO ESCs and NF90 + NF110 KO ESCs show similar expression changes. Moreover, NF90 + NF110 RNA immunoprecipitation (RIP)-seq in ESCs suggested that NF90/NF110 directly regulate proliferation, differentiation, and RNA-processing genes. Our data support a model in which NF45, NF90, and NF110 operate in feedback loops that enable them, through both overlapping and independent targets, to help balance the push and pull of pluripotency and differentiation cues.

Regulation of cell cycle of hepatocellular carcinoma by NF90 through modulation of cyclin E1 mRNA stability.

Activation of cyclin E1, a key regulator of the G1/S cell-cycle transition, has been implicated in many cancers including hepatocellular carcinoma (HCC). Although much is known about the regulation of cyclin E1 expression and stability, its post-transcriptional regulation mechanism remains incompletely understood. Here, we report that nuclear factor 90 (NF90), a double-stranded RNA (dsRNA) binding protein, regulates cyclin E1 in HCC. We demonstrate that NF90 is upregulated in HCC specimens and that suppression of NF90 decreases HCC cell growth and delays G1/S transition. We identified cyclin E1 as a new target of NF90 and found a significant correlation between NF90 and cyclin E1 expression in HCC. The mRNA and protein levels of cyclin E1 were downregulated upon NF90 knockdown. Suppression of NF90 caused a decrease in the half-life of cyclin E1 mRNA, which was rescued by ectopic expression of NF90. Furthermore, NF90 bound to the 3' untranslated regions (3'UTRs) of cyclin E1 mRNA in vitro and in vivo. Knockdown of NF90 also inhibited tumor growth of HCC cell lines in mouse xenograft model. Moreover, we showed that inhibition of NF90 sensitized HCC cells to the cyclin-dependent kinase 2 (CDK2) inhibitor, roscovitine. Taken together, downregulation of NF90 in HCC cell lines can delay cell-cycle progression, inhibit cell proliferation, and reduce tumorigenic capacity in vivo. These results suggest that NF90 has an important role in HCC pathogenesis and that it can serve as a novel therapeutic target for HCC.

hnRNP L and NF90 interact with hepatitis C virus 5'-terminal untranslated RNA and promote efficient replication.

UNLABELLED: The 5'-terminal sequence of the hepatitis C virus (HCV) positive-strand RNA genome is essential for viral replication. Critical host factors, including a miR-122/Ago2 complex and poly(rC)-binding protein 2 (PCBP2), associate with this RNA segment. We used a biotinylated RNA pulldown approach to isolate host factors binding to the HCV 5' terminal 47 nucleotides and, in addition to Ago2 and PCBP2, identified several novel proteins, including IGF2BP1, hnRNP L, DHX9, ADAR1, and NF90 (ILF3). PCBP2, IGF2BP1, and hnRNP L bound single-stranded RNA, while DHX9, ADAR1, and NF90 bound a cognate double-stranded RNA bait. PCBP2, IGF2BP1, and hnRNP L binding were blocked by preannealing the single-stranded RNA bait with miR-122, indicating that they bind the RNA in competition with miR-122. However, IGF2BP1 binding was also inhibited by high concentrations of heparin, suggesting that it bound the bait nonspecifically. Among these proteins, small interfering RNA-mediated depletion of hnRNP L and NF90 significantly impaired viral replication and reduced infectious virus yields without substantially affecting HCV internal ribosome entry site-mediated translation. hnRNP L and NF90 were found to associate with HCV RNA in infected cells and to coimmunoprecipitate with NS5A in an RNA-dependent manner. Both also associate with detergent-resistant membranes where viral replication complexes reside. We conclude that hnRNP and NF90 are important host factors for HCV replication, at least in cultured cells, and may be present in the replication complex. IMPORTANCE: Although HCV replication has been intensively studied in many laboratories, many aspects of the viral life cycle remain obscure. Here, we use a novel RNA pulldown strategy coupled with mass spectrometry to identify host cell proteins that interact functionally with regulatory RNA elements located at the extreme 5' end of the positive-strand RNA genome. We identify two, primarily nuclear RNA-binding proteins, hnRNP L and NF90, with previously unrecognized proviral roles in HCV replication. The data presented add to current understanding of the replication cycle of this pathogenic human virus.

Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon.

Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and ß chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the 'MHCII RNA operon' in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5'- and 3'-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.

Sepantronium bromide (YM155) induces disruption of the ILF3/p54(nrb) complex, which is required for survivin expression.

YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin[1]. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.

EBP1 and DRBP76/NF90 binding proteins are included in the major histocompatibility complex class II RNA operon.

Major histocompatibility complex class II mRNAs encode heterodimeric proteins involved in the presentation of exogenous antigens during an immune response. Their 3'UTRs bind a protein complex in which we identified two factors: EBP1, an ErbB3 receptor-binding protein and DRBP76, a double-stranded RNA binding nuclear protein, also known as nuclear factor 90 (NF90). Both are well-characterized regulatory factors of several mRNA molecules processing. Using either EBP1 or DRBP76/NF90-specific knockdown experiments, we established that the two proteins play a role in regulating the expression of HLA-DRA, HLA-DRB1 and HLA-DQA1 mRNAs levels. Our study represents the first indication of the existence of a functional unit that includes different transcripts involved in the adaptive immune response. We propose that the concept of 'RNA operon' may be suitable for our system in which MHCII mRNAs are modulated via interaction of their 3'UTR with same proteins.

NF90 binds the dengue virus RNA 3' terminus and is a positive regulator of dengue virus replication.

BACKGROUND: Viral RNA translation and replication are regulated by sequence and structural elements in the 5' and 3' untranslated regions (UTR) and by host cell and/or viral proteins that bind them. Dengue virus has a single-stranded RNA genome with positive polarity, a 5' m7GpppG cap, and a conserved 3'-terminal stem loop (SL) that is linked to proposed functions in viral RNA transcription and translation. Mechanisms explaining the contributions of host proteins to viral RNA translation and replication are poorly defined, yet understanding host protein-viral RNA interactions may identify new targets for therapeutic intervention. This study was directed at identifying functionally significant host proteins that bind the conserved dengue virus RNA 3' terminus. METHODOLOGY/PRINCIPAL FINDINGS: Proteins eluted from a dengue 3' SL RNA affinity column at increasing ionic strength included two with double-strand RNA binding motifs (NF90/DRBP76 and DEAH box polypeptide 9/RNA helicase A (RHA)), in addition to NF45, which forms a heterodimer with NF90. Although detectable NF90 and RHA proteins localized to the nucleus of uninfected cells, immunofluorescence revealed cytoplasmic NF90 in dengue virus-infected cells, leading us to hypothesize that NF90 has a functional role(s) in dengue infections. Cells depleted of NF90 were used to quantify viral RNA transcript levels and production of infectious dengue virus. NF90 depletion was accompanied by a 50%-70% decrease in dengue RNA levels and in production of infectious viral progeny. CONCLUSIONS/SIGNIFICANCE: The results indicate that NF90 interacts with the 3' SL structure of the dengue RNA and is a positive regulator of dengue virus replication. NF90 depletion diminished the production of infectious dengue virus by more than 50%, which may have important significance for identifying therapeutic targets to limit a virus that threatens more than a billion people worldwide.

NF90 selectively represses the translation of target mRNAs bearing an AU-rich signature motif.

The RNA-binding protein nuclear factor 90 (NF90) has been implicated in the stabilization, transport and translational control of several target mRNAs. However, a systematic analysis of NF90 target mRNAs has not been performed. Here, we use ribonucleoprotein immunoprecipitation analysis to identify a large subset of NF90-associated mRNAs. Comparison of the 3'-untranslated regions (UTRs) of these mRNAs led to the elucidation of a 25- to 30-nucleotide, RNA signature motif rich in adenines and uracils. Insertion of the AU-rich NF90 motif ('NF90m') in the 3'UTR of an EGFP heterologous reporter did not affect the steady-state level of the chimeric EGFP-NF90m mRNA or its cytosolic abundance. Instead, the translation of EGFP-NF90m mRNA was specifically repressed in an NF90-dependent manner, as determined by analysing nascent EGFP translation, the distribution of chimeric mRNAs on polysome gradients and the steady-state levels of expressed EGFP protein. The interaction of endogenous NF90 with target mRNAs was validated after testing both endogenous mRNAs and recombinant biotinylated transcripts containing NF90 motif hits. Further analysis showed that the stability of endogenous NF90 target mRNAs was not significantly influenced by NF90 abundance, while their translation increased when NF90 levels were reduced. In summary, we have identified an AU-rich RNA motif present in NF90 target mRNAs and have obtained evidence that NF90 represses the translation of this subset of mRNAs.