PPM1D suppresses p53-dependent transactivation and cell death by inhibiting the Integrated Stress Response.

The p53 transcription factor is a master regulator of cellular stress responses inhibited by repressors such as MDM2 and the phosphatase PPM1D. Activation of p53 with pharmacological inhibitors of its repressors is being tested in clinical trials for cancer therapy, but efficacy has been limited by poor induction of tumor cell death. We demonstrate that dual inhibition of MDM2 and PPM1D induces apoptosis in multiple cancer cell types via amplification of the p53 transcriptional program through the eIF2α-ATF4 pathway. PPM1D inhibition induces phosphorylation of eIF2α, ATF4 accumulation, and ATF4-dependent enhancement of p53-dependent transactivation upon MDM2 inhibition. Dual inhibition of p53 repressors depletes heme and induces HRI-dependent eIF2α phosphorylation. Pharmacological induction of eIF2α phosphorylation synergizes with MDM2 inhibition to induce cell death and halt tumor growth in mice. These results demonstrate that PPM1D inhibits both the p53 network and the integrated stress response controlled by eIF2α-ATF4, with clear therapeutic implications.

Biomotors, viral assembly, and RNA nanobiotechnology: Current achievements and future directions.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure-function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields.

Therapeutic resistance in acute myeloid leukemia cells is mediated by a novel ATM/mTOR pathway regulating oxidative phosphorylation.

While leukemic cells are susceptible to various therapeutic insults, residence in the bone marrow microenvironment typically confers protection from a wide range of drugs. Thus, understanding the unique molecular changes elicited by the marrow is of critical importance toward improving therapeutic outcomes. In this study, we demonstrate that aberrant activation of oxidative phosphorylation serves to induce therapeutic resistance in FLT3 mutant human AML cells challenged with FLT3 inhibitor drugs. Importantly, our findings show that AML cells are protected from apoptosis following FLT3 inhibition due to marrow-mediated activation of ATM, which in turn upregulates oxidative phosphorylation via mTOR signaling. mTOR is required for the bone marrow stroma-dependent maintenance of protein translation, with selective polysome enrichment of oxidative phosphorylation transcripts, despite FLT3 inhibition. To investigate the therapeutic significance of this finding, we tested the mTOR inhibitor everolimus in combination with the FLT3 inhibitor quizartinib in primary human AML xenograft models. While marrow resident AML cells were highly resistant to quizartinib alone, the addition of everolimus induced profound reduction in tumor burden and prevented relapse. Taken together, these data provide a novel mechanistic understanding of marrow-based therapeutic resistance and a promising strategy for improved treatment of FLT3 mutant AML patients.

Integrated stress response control of granulosa cell translation and proliferation during normal ovarian follicle development.

Mechanisms that directly control mammalian ovarian primordial follicle (PF) growth activation and the selection of individual follicles for survival are largely unknown. Follicle cells produce factors that can act as potent inducers of cellular stress during normal function. Consistent with this, we show here that normal, untreated ovarian cells, including pre-granulosa cells of dormant PFs, express phenotype and protein markers of the activated integrated stress response (ISR), including stress-specific protein translation (phospho-Serine 51 eukaryotic initiation factor 2α; P-EIF2α), active DNA damage checkpoints, and cell-cycle arrest. We further demonstrate that mRNAs upregulated in primary (growing) follicles versus arrested PFs mostly include stress-responsive upstream open reading frames (uORFs). Treatment of a granulosa cell (GC) line with the PF growth trigger tumor necrosis factor alpha results in the upregulation of a 'stress-dependent' translation profile. This includes further elevated P-eIF2α and a shift of uORF-containing mRNAs to polysomes. Because the active ISR corresponds to slow follicle growth and PF arrest, we propose that repair and abrogation of ISR checkpoints (e.g. checkpoint recovery) drives the GC cell cycle and PF growth activation (PFGA). If cellular stress is elevated beyond a threshold(s) or, if damage occurs that cannot be repaired, cell and follicle death ensue, consistent with physiological atresia. These data suggest an intrinsic quality control mechanism for immature and growing follicles, where PFGA and subsequent follicle growth and survival depend causally upon ISR resolution, including DNA repair and thus the proof of genomic integrity.

Recognition of non-CpG repeats in Alu and ribosomal RNAs by the Z-RNA binding domain of ADAR1 induces A-Z junctions.

Adenosine-to-inosine (A-to-I) editing of eukaryotic cellular RNAs is essential for protection against auto-immune disorders. Editing is carried out by ADAR1, whose innate immune response-specific cytoplasmic isoform possesses a Z-DNA binding domain (Zα) of unknown function. Zα also binds to CpG repeats in RNA, which are a hallmark of Z-RNA formation. Unexpectedly, Zα has been predicted - and in some cases even shown - to bind to specific regions within mRNA and rRNA devoid of such repeats. Here, we use NMR, circular dichroism, and other biophysical approaches to demonstrate and characterize the binding of Zα to mRNA and rRNA fragments. Our results reveal a broad range of RNA sequences that bind to Zα and adopt Z-RNA conformations. Binding is accompanied by destabilization of neighboring A-form regions which is similar in character to what has been observed for B-Z-DNA junctions. The binding of Zα to non-CpG sequences is specific, cooperative and occurs with an affinity in the low micromolar range. This work allows us to propose a model for how Zα could influence the RNA binding specificity of ADAR1.

An RNA pseudoknot stimulates HTLV-1 pro-pol programmed -1 ribosomal frameshifting.

Programmed -1 ribosomal frameshifts (-1 PRFs) are commonly used by viruses to regulate their enzymatic and structural protein levels. Human T-cell leukemia virus type 1 (HTLV-1) is a carcinogenic retrovirus that uses two independent -1 PRFs to express viral enzymes critical to establishing new HTLV-1 infections. How the cis-acting RNA elements in this viral transcript function to induce frameshifting is unknown. The objective of this work was to conclusively define the 3' boundary of and the RNA elements within the HTLV-1 pro-pol frameshift site. We hypothesized that the frameshift site structure was a pseudoknot and that its 3' boundary would be defined by the pseudoknot's 3' end. To test these hypotheses, the in vitro frameshift efficiencies of three HTLV-1 pro-pol frameshift sites with different 3' boundaries were quantified. The results indicated that nucleotides included in the longest construct were essential to highly efficient frameshift stimulation. Interestingly, only this construct could form the putative frameshift site pseudoknot. Next, the secondary structure of this frameshift site was determined. The dominant structure was an H-type pseudoknot which, together with the slippery sequence, stimulated frameshifting to 19.4(±0.3)%. The pseudoknot's critical role in frameshift stimulation was directly revealed by examining the impact of structural changes on HTLV-1 pro-pol -1 PRF. As predicted, mutations that occluded pseudoknot formation drastically reduced the frameshift efficiency. These results are significant because they demonstrate that a pseudoknot is important to HTLV-1 pro-pol -1 PRF and define the frameshift site's 3' boundary.

Zika virus produces noncoding RNAs using a multi-pseudoknot structure that confounds a cellular exonuclease.

The outbreak of Zika virus (ZIKV) and associated fetal microcephaly mandates efforts to understand the molecular processes of infection. Related flaviviruses produce noncoding subgenomic flaviviral RNAs (sfRNAs) that are linked to pathogenicity in fetal mice. These viruses make sfRNAs by co-opting a cellular exonuclease via structured RNAs called xrRNAs. We found that ZIKV-infected monkey and human epithelial cells, mouse neurons, and mosquito cells produce sfRNAs. The RNA structure that is responsible for ZIKV sfRNA production forms a complex fold that is likely found in many pathogenic flaviviruses. Mutations that disrupt the structure affect exonuclease resistance in vitro and sfRNA formation during infection. The complete ZIKV xrRNA structure clarifies the mechanism of exonuclease resistance and identifies features that may modulate function in diverse flaviviruses.

Structured RNAs that evade or confound exonucleases: function follows form.

Cells contain powerful RNA decay machinery to eliminate unneeded RNA from the cell, and this process is an important and regulated part of controlling gene expression. However, certain structured RNAs have been found that can robustly resist degradation and extend the lifetime of an RNA. In this review, we present three RNA structures that use a specific three-dimensional fold to provide protection from RNA degradation, and discuss how the recently-solved structures of these RNAs explain their function. Specifically, we describe the Xrn1-resistant RNAs from arthropod-borne flaviviruses, exosome-resistant long non-coding RNAs associated with lung cancer metastasis and found in Kaposi's sarcoma-associated herpesvirus, and tRNA-like sequences occurring in certain plant viruses. These three structures reveal three different mechanisms to protect RNAs from decay and suggest RNA structure-based nuclease resistance may be a widespread mechanism of regulation.

Structural basis of chronic beryllium disease: linking allergic hypersensitivity and autoimmunity.

T-cell-mediated hypersensitivity to metal cations is common in humans. How the T cell antigen receptor (TCR) recognizes these cations bound to a major histocompatibility complex (MHC) protein and self-peptide is unknown. Individuals carrying the MHCII allele, HLA-DP2, are at risk for chronic beryllium disease (CBD), a debilitating inflammatory lung condition caused by the reaction of CD4 T cells to inhaled beryllium. Here, we show that the T cell ligand is created when a Be(2+) cation becomes buried in an HLA-DP2/peptide complex, where it is coordinated by both MHC and peptide acidic amino acids. Surprisingly, the TCR does not interact with the Be(2+) itself, but rather with surface changes induced by the firmly bound Be(2+) and an accompanying Na(+) cation. Thus, CBD, by creating a new antigen by indirectly modifying the structure of preexisting self MHC-peptide complex, lies on the border between allergic hypersensitivity and autoimmunity.

Translation initiation factor eIF3b expression in human cancer and its role in tumor growth and lung colonization.

PURPOSE: Discovery transcriptomic analyses suggest eukaryotic initiation factor 3b (eIF3b) is elevated in human bladder and prostate cancer, yet its role as a prognostic factor or its requirement in the maintenance or progression of human cancer is not established. Here, we determine the therapeutic potential of eIF3b by examining the clinical relevance of its expression in human cancer tissues and its role in experimental tumor models. EXPERIMENTAL DESIGN: We examined mRNA expression of eIF3b in bladder (N = 317) and prostate (N = 566) tissue samples and protein expression by immunohistochemistry in 143 bladder tumor samples as a function of clinicopathologic features. The impact of eIF3b depletion by siRNA in human cancer lines was evaluated in regard to in vitro cell growth, cell cycle, migration, in vivo subcutaneous tumor growth, and lung colonization. RESULTS: eIF3b mRNA expression correlated to tumor grade, stage, and survival in human bladder and prostate cancer. eIF3b protein expression stratified survival in human bladder cancer. eIF3b depletion reduced in vitro cancer cell growth; inhibited G1-S cell-cycle transition by changing protein but not RNA expression of cyclin A, E, Rb, and p27Kip1; inhibited migration; and disrupted actin cytoskeleton and focal adhesions. These changes were associated with decreased protein expression of integrin α5. Integrin α5 depletion phenocopied effects observed with eIF3b. eIF3b-depleted bladder cancer cells formed fewer subcutaneous tumors that grew more slowly and had reduced lung colonization. CONCLUSION: eIF3b expression relates to human bladder and prostate cancer prognosis, is required for tumor growth, and thus a candidate therapeutic target.

Structural basis for nematode eIF4E binding an m(2,2,7)G-Cap and its implications for translation initiation.

Metazoan spliced leader (SL) trans-splicing generates mRNAs with an m(2,2,7)G-cap and a common downstream SL RNA sequence. The mechanism for eIF4E binding an m²²7G-cap is unknown. Here, we describe the first structure of an eIF4E with an m(2,2,7)G-cap and compare it to the cognate m7G-eIF4E complex. These structures and Nuclear Magnetic Resonance (NMR) data indicate that the nematode Ascaris suum eIF4E binds the two different caps in a similar manner except for the loss of a single hydrogen bond on binding the m(2,2,7)G-cap. Nematode and mammalian eIF4E both have a low affinity for m(2,2,7)G-cap compared with the m7G-cap. Nematode eIF4E binding to the m7G-cap, m(2,2,7)G-cap and the m(2,2,7)G-SL 22-nt RNA leads to distinct eIF4E conformational changes. Additional interactions occur between Ascaris eIF4E and the SL on binding the m(2,2,7)G-SL. We propose interactions between Ascaris eIF4E and the SL impact eIF4G and contribute to translation initiation, whereas these interactions do not occur when only the m(2,2,7)G-cap is present. These data have implications for the contribution of 5'-UTRs in mRNA translation and the function of different eIF4E isoforms.

The retinal specific CD147 Ig0 domain: from molecular structure to biological activity.

CD147 is a type I transmembrane protein that is involved in inflammatory diseases, cancer progression, and multiple human pathogens utilize CD147 for efficient infection. CD147 expression is so high in several cancers that it is now used as a prognostic marker. The two primary isoforms of CD147 that are related to cancer progression have been identified, differing in their number of immunoglobulin (Ig)-like domains. These include CD147 Ig1-Ig2, which is ubiquitously expressed in most tissues, and CD147 Ig0-Ig1-Ig2, which is retinal specific and implicated in retinoblastoma. However, little is known in regard to the retinal specific CD147 Ig0 domain despite its potential role in retinoblastoma. We present the first crystal structure of the human CD147 Ig0 domain and show that the CD147 Ig0 domain is a crystallographic dimer with an I-type domain structure, which maintained in solution. Furthermore, we have utilized our structural data together with mutagenesis to probe the biological activity of CD147-containing proteins, both with and without the CD147 Ig0 domain, within several model cell lines. Our findings reveal that the CD147 Ig0 domain is a potent stimulator of interleukin-6 and suggest that the CD147 Ig0 domain has its own receptor distinct from that of the other CD147 Ig-like domains, CD147 Ig1-Ig2. Finally, we show that the CD147 Ig0 dimer is the functional unit required for activity and can be disrupted by a single point mutation.

A synonymous variant in scavenger receptor, class B, type I gene is associated with lower SR-BI protein expression and function.

OBJECTIVE: A synonymous variant within scavenger receptor class B type I gene (SCARB1), exon 8 rs5888, has been associated with altered lipid levels and cardiovascular risk in humans. The objective was to determine if rs5888 decreased SR-BI protein expression and function in vitro. METHODS: SR-BI RNA secondary structure, turnover, polysomal distribution and protein expression were examined in COS cells transfected with wild-type or rs5888-SR-BI plasmids by selective 2'-hydroxyl acylation and primer extension assays, actinomycin D inhibition, polysomal profiling, and western blotting. SR-BI function in murine macrophages stably expressing wild-type or rs5888-SR-BI was assessed by measuring the specific cell association of (125)I,(3)H-cholesteryl ester (CE) radiolabeled HDL. RESULTS: Rs5888 changed RNA secondary structure and led to marked differences in the polysomal profiles compared with wild-type transcript (p<0.02). As compared to wild-type cells, COS cells expressing rs5888 had significantly lower SR-BI protein expression (p<0.04), but no difference in total RNA transcript levels. There were no differences in SR-BI RNA turnover in murine macrophages, whereas specific cell association of (125)I (p<0.0001) or (3)H-CE (p<0.00001) was significantly lower in rs5888 cells. CONCLUSIONS: The rs5888 variant affected SR-BI RNA secondary structure, protein translation, and was significantly associated with reduced SR-BI protein expression and function in vitro.

An approach to the construction of tailor-made amphiphilic peptides that strongly and selectively bind to hairpin RNA targets.

The hairpin RNA motif is one of the most frequently observed secondary structures and is often targeted by therapeutic agents. An amphiphilic peptide with seven lysine and eight leucine residues and its derivatives were designed for use as ligands against RNA hairpin motifs. We hypothesized that variations in both the hydrophobic leucine-rich and hydrophilic lysine-rich spheres of these amphiphilic peptides would create extra attractive interactions with hairpin RNA targets. A series of alanine-scanned peptides were probed to identify the most influential lysine residues in the hydrophilic sphere. The binding affinities of these modified peptides with several hairpins, such as RRE, TAR from HIV, a short hairpin from IRES of HCV, and a hairpin from the 16S A-site stem from rRNA, were determined. Since the hairpin from IRES of HCV was the most susceptible to the initial series of alanine-scanned peptides, studies investigating how further variations in the peptides effect binding employed the IRES hairpin. Next, the important Lys residues were substituted by shorter chain amines, such as ornithine, to place the peptide deeper into the hairpin groove. In a few cases, a 70-fold improved binding was observed for peptides that contained the specifically located shorter amine side chains. To further explore changes in binding affinities brought about by alterations in the hydrophobic sphere, tryptophan residues were introduced in place of leucine. A few peptides with tryptophan in specific positions also displayed 70-fold improved binding affinities. Finally, double mutant peptides incorporating both specifically located shorter amine side chains in the hydrophilic region and tryptophan residues in the hydrophobic region were synthesized. The binding affinities of peptides containing the simple double modification were observed to be 80 times lower, and their binding specificities were increased 40-fold. The results of this effort provide important information about strategies that can be used to prepare peptides that both strongly and selectively target hairpin RNAs. Specifically, the findings indicate that tailor-made amphiphilic peptide ligands against certain hairpin RNAs can be obtained if the RNA target possesses a deep groove in which both the hydrophobic and hydrophilic spheres of the peptide interact.

Comparison and functional implications of the 3D architectures of viral tRNA-like structures.

RNA viruses co-opt the host cell's biological machinery, and their infection strategies often depend on specific structures in the viral genomic RNA. Examples are tRNA-like structures (TLSs), found at the 3' end of certain plant viral RNAs, which can use the cell's aminoacyl tRNA-synthetases (AARSs) to drive addition of an amino acid to the 3' end of the viral RNA. TLSs are multifunctional RNAs involved in processes such as viral replication, translation, and viral RNA stability; these functions depend on their fold. Experimental result-based structural models of TLSs have been published. In this study, we further examine these structures using a combination of biophysical and biochemical approaches to explore the three-dimensional (3D) architectures of TLSs from the turnip yellow mosaic virus (TYMV), tobacco mosaic virus (TMV), and brome mosaic virus (BMV). We find that despite similar function, these RNAs are biophysically diverse: the TYMV TLS adopts a characteristic tRNA-like L shape, the BMV TLS has a large compact globular domain with several helical extensions, and the TMV TLS aggregates in solution. Both the TYMV and BMV TLS RNAs adopt structures with tight backbone packing and also with dynamic structural elements, suggesting complexities and subtleties that cannot be explained by simple tRNA mimicry. These results confirm some aspects of existing models and also indicate how these models can be improved. The biophysical characteristics of these TLSs show how these multifunctional RNAs might regulate various viral processes, including negative strand synthesis, and also allow comparison with other structured RNAs.

The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.