MIR-NATs repress MAPT translation and aid proteostasis in neurodegeneration.

The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes1,2. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site3. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies4. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies5 and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs6, with particular relevance for proteostasis in neurodegeneration.

C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation.

Intronic GGGGCC (G4C2) hexanucleotide repeat expansion within the human C9orf72 gene represents the most common cause of familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9ALS/FTD). Repeat-associated non-AUG (RAN) translation of repeat-containing C9orf72 RNA results in the production of neurotoxic dipeptide-repeat proteins (DPRs). Here, we developed a high-throughput drug screen for the identification of positive and negative modulators of DPR levels. We found that HSP90 inhibitor geldanamycin and aldosterone antagonist spironolactone reduced DPR levels by promoting protein degradation via the proteasome and autophagy pathways respectively. Surprisingly, cAMP-elevating compounds boosting protein kinase A (PKA) activity increased DPR levels. Inhibition of PKA activity, by both pharmacological and genetic approaches, reduced DPR levels in cells and rescued pathological phenotypes in a Drosophila model of C9ALS/FTD. Moreover, knockdown of PKA-catalytic subunits correlated with reduced translation efficiency of DPRs, while the PKA inhibitor H89 reduced endogenous DPR levels in C9ALS/FTD patient-derived iPSC motor neurons. Together, our results suggest new and druggable pathways modulating DPR levels in C9ALS/FTD.

HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA.

The RNA-binding protein HuD promotes neurogenesis and favors recovery from peripheral axon injury. HuD interacts with many mRNAs, altering both stability and translation efficiency. We generated a nucleotide resolution map of the HuD RNA interactome in motor neuron-like cells, identifying HuD target sites in 1,304 mRNAs, almost exclusively in the 3' UTR. HuD binds many mRNAs encoding mTORC1-responsive ribosomal proteins and translation factors. Altered HuD expression correlates with the translation efficiency of these mRNAs and overall protein synthesis, in a mTORC1-independent fashion. The predominant HuD target is the abundant, small non-coding RNA Y3, amounting to 70% of the HuD interaction signal. Y3 functions as a molecular sponge for HuD, dynamically limiting its recruitment to polysomes and its activity as a translation and neuron differentiation enhancer. These findings uncover an alternative route to the mTORC1 pathway for translational control in motor neurons that is tunable by a small non-coding RNA.

Ythdf is a N6-methyladenosine reader that modulates Fmr1 target mRNA selection and restricts axonal growth in Drosophila.

N6-methyladenosine (m6 A) regulates a variety of physiological processes through modulation of RNA metabolism. This modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6 A alters fly behavior, albeit the underlying molecular mechanism and the role of m6 A during nervous system development have remained elusive. Here we find that impairment of the m6 A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6 A reader in the nervous system, being required to limit axonal growth. Mechanistically, we show that the m6 A reader Ythdf directly interacts with Fmr1, the fly homolog of Fragile X mental retardation RNA binding protein (FMRP), to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6 A pathway controls development of the nervous system and modulates Fmr1 target transcript selection.

From the identification of actionable molecular targets to the generation of faithful neuroblastoma patient-derived preclinical models.

BACKGROUND: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Although the overall survival of patients with NB has improved in the last years, more than 50% of high-risk patients still undergo a relapse. Thus, in the era of precision/personalized medicine, the need for high-risk NB patient-specific therapies is urgent. METHODS: Within the PeRsonalizEd Medicine (PREME) program, patient-derived NB tumors and bone marrow (BM)-infiltrating NB cells, derived from either iliac crests or tumor bone lesions, underwent to histological and to flow cytometry immunophenotyping, respectively. BM samples containing a NB cells infiltration from 1 to 50 percent, underwent to a subsequent NB cells enrichment using immune-magnetic manipulation. Then, NB samples were used for the identification of actionable targets and for the generation of 3D/tumor-spheres and Patient-Derived Xenografts (PDX) and Cell PDX (CPDX) preclinical models. RESULTS: Eighty-four percent of NB-patients showed potentially therapeutically targetable somatic alterations (including point mutations, copy number variations and mRNA over-expression). Sixty-six percent of samples showed alterations, graded as "very high priority", that are validated to be directly targetable by an approved drug or an investigational agent. A molecular targeted therapy was applied for four patients, while a genetic counseling was suggested to two patients having one pathogenic germline variant in known cancer predisposition genes. Out of eleven samples implanted in mice, five gave rise to (C)PDX, all preserved in a local PDX Bio-bank. Interestingly, comparing all molecular alterations and histological and immunophenotypic features among the original patient's tumors and PDX/CPDX up to second generation, a high grade of similarity was observed. Notably, also 3D models conserved immunophenotypic features and molecular alterations of the original tumors. CONCLUSIONS: PREME confirms the possibility of identifying targetable genomic alterations in NB, indeed, a molecular targeted therapy was applied to four NB patients. PREME paves the way to the creation of clinically relevant repositories of faithful patient-derived (C)PDX and 3D models, on which testing precision, NB standard-of-care and experimental medicines.

Transient rapamycin treatment during developmental stage extends lifespan in Mus musculus and Drosophila melanogaster.

Lifespan is determined by complex and tangled mechanisms that are largely unknown. The early postnatal stage has been proposed to play a role in lifespan, but its contribution is still controversial. Here, we show that a short rapamycin treatment during early life can prolong lifespan in Mus musculus and Drosophila melanogaster. Notably, the same treatment at later time points has no effect on lifespan, suggesting that a specific time window is involved in lifespan regulation. We also find that sulfotransferases are upregulated during early rapamycin treatment both in newborn mice and in Drosophila larvae, and transient dST1 overexpression in Drosophila larvae extends lifespan. Our findings unveil a novel link between early-life treatments and long-term effects on lifespan.

Pliability in the m6A-Binding Region Extends Druggability of YTH Domains.

Epitranscriptomic mRNA modifications affect gene expression, with their altered balance detected in various cancers. YTHDF proteins contain the YTH reader domain recognizing the m6A mark on mRNA and represent valuable drug targets. Crystallographic structures have been determined for all three family members; however, discrepancies are present in the organization of the m6A-binding pocket. Here, we present new crystallographic structures of the YTH domain of YTHDF1, accompanied by computational studies, showing that this domain can exist in different stable conformations separated by a significant energetic barrier. During the transition, additional conformations are explored, with peculiar druggable pockets appearing and offering new opportunities for the design of YTH-interfering small molecules.

Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency.

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.

EGCG Disrupts the LIN28B/Let-7 Interaction and Reduces Neuroblastoma Aggressiveness.

Neuroblastoma (NB) is the most commonly diagnosed extracranial solid tumor in children, accounting for 15% of all childhood cancer deaths. Although the 5-year survival rate of patients with a high-risk disease has increased in recent decades, NB remains a challenge in pediatric oncology, and the identification of novel potential therapeutic targets and agents is an urgent clinical need. The RNA-binding protein LIN28B has been identified as an oncogene in NB and is associated with a poor prognosis. Given that LIN28B acts by negatively regulating the biogenesis of the tumor suppressor let-7 miRNAs, we reasoned that selective interference with the LIN28B/let-7 miRNA interaction would increase let-7 miRNA levels, ultimately leading to reduced NB aggressiveness. Here, we selected (-)-epigallocatechin 3-gallate (EGCG) out of 4959 molecules screened as the molecule with the best inhibitory activity on LIN28B/let-7 miRNA interaction and showed that treatment with PLC/PLGA-PEG nanoparticles containing EGCG (EGCG-NPs) led to an increase in mature let-7 miRNAs and a consequent inhibition of NB cell growth. In addition, EGCG-NP pretreatment reduced the tumorigenic potential of NB cells in vivo. These experiments suggest that the LIN28B/let-7 miRNA axis is a good therapeutic target in NB and that EGCG, which can interfere with this interaction, deserves further preclinical evaluation.

Expanding the Chemical Space of Arsenicin A-C Related Polyarsenicals and Evaluation of Some Analogs as Inhibitors of Glioblastoma Stem Cell Growth.

The marine polyarsenical metabolite arsenicin A is the landmark of a series of natural and synthetic molecules characterized by an adamantane-like tetraarsenic cage. Arsenicin A and related polyarsenicals have been evaluated for their antitumor effects in vitro and have been proven more potent than the FDA-approved arsenic trioxide. In this context, we have expanded the chemical space of polyarsenicals related to arsenicin A by synthesizing dialkyl and dimethyl thio-analogs, the latter characterized with the support of simulated NMR spectra. In addition, the new natural arsenicin D, the scarcity of which in the Echinochalina bargibanti extract had previously limited its full structural characterization, has been identified by synthesis. The dialkyl analogs, which present the adamantane-like arsenicin A cage substituted with either two methyl, ethyl, or propyl chains, were efficiently and selectively produced and evaluated for their activity on glioblastoma stem cells (GSCs), a promising therapeutic target in glioblastoma treatment. These compounds inhibited the growth of nine GSC lines more potently than arsenic trioxide, with GI50 values in the submicromolar range, both under normoxic and hypoxic conditions, and presented high selectivity toward non-tumor cell lines. The diethyl and dipropyl analogs, which present favorable physical-chemical and ADME parameters, had the most promising results.

Hypomorphic mutation of the mouse Huntington's disease gene orthologue.

Rare individuals with inactivating mutations in the Huntington's disease gene (HTT) exhibit variable abnormalities that imply essential HTT roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in the murine HTT orthologue Htt, (HdhneoQ20, HdhneoQ50, HdhneoQ111), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate, though still severe, alleles yielded recessive perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development that nominate polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that Htt is required both pre- and post-gastrulation to support normal development.

Association of PARP1 polymorphisms with response to chemotherapy in patients with high-risk neuroblastoma.

The genetic aetiology and the molecular mechanisms that characterize high-risk neuroblastoma are still little understood. The majority of high-risk neuroblastoma patients do not take advantage of current induction therapy. So far, one of the main reasons liable for cancer therapeutic failure is the acquisition of resistance to cytotoxic anticancer drugs, because of the DNA repair system of tumour cells. PARP1 is one of the main DNA damage sensors involved in the DNA repair system and genomic stability. We observed that high PARP1 mRNA level is associated with unfavourable prognosis in 3 public gene expression NB patients' datasets and in 20 neuroblastomas analysed by qRT-PCR. Among 4983 SNPs in PARP1, we selected two potential functional SNPs. We investigated the association of rs907187, in PARP1 promoter, and rs2048426 in non-coding region with response chemotherapy in 121 Italian patients with high-risk NB. Results showed that minor G allele of rs907187 associated with induction response of patients (P = .02) and with decrease PARP1 mRNA levels in NB cell line (P = .003). Furthermore, rs907187 was predicted to alter the binding site of E2F1 transcription factor. Specifically, allele G had low binding affinity with E2F1 whose expression positively correlates with PARP1 expression and associated with poor prognosis of patients with NB. By contrast, we did not find genetic association for the SNP rs2048426. These data reveal rs907187 as a novel potential risk variant associated with the failure of induction therapy for high-risk NB.

Hydrogen peroxide is a neuronal alarmin that triggers specific RNAs, local translation of Annexin A2, and cytoskeletal remodeling in Schwann cells.

Schwann cells are key players in neuro-regeneration: They sense "alarm" signals released by degenerating nerve terminals and differentiate toward a proregenerative phenotype, with phagocytosis of nerve debris and nerve guidance. At the murine neuromuscular junction, hydrogen peroxide (H2O2) is a key signal of Schwann cells' activation in response to a variety of nerve injuries. Here we report that Schwann cells exposed to low doses of H2O2 rewire the expression of several RNAs at both transcriptional and translational levels. Among the genes positively regulated at both levels, we identified an enriched cluster involved in cytoskeleton remodeling and cell migration, with the Annexin (Anxa) proteins being the most represented family. We show that both Annexin A2 (Anxa2) transcript and protein accumulate at the tips of long pseudopods that Schwann cells extend upon H2O2 exposure. Interestingly, Schwann cells reply to this signal and to nerve injury by locally translating Anxa2 in pseudopods, and undergo an extensive cytoskeleton remodeling. Our results show that, similarly to neurons, Schwann cells take advantage of local protein synthesis to change shape and move toward damaged axonal terminals to facilitate axonal regeneration.

Co-Administration of Fendiline Hydrochloride Enhances Chemotherapeutic Efficacy of Cisplatin in Neuroblastoma Treatment.

Despite significant improvement of neuroblastoma (NB) patients' survival due to recent treatment advancements in recent years, NB is still associated with high mortality rate. In search of novel strategies to increase NB's susceptibility to pharmacological treatments, we investigated the in vitro and in vivo effects of fendiline hydrochloride as an enhancer of cisplatin antitumor activity. To assess the modulation of fendiline treatment on cisplatin responses, we used in vitro (evaluating NB cell proliferation by XCELLigence technology and colony formation, and gene expression by RT-PCR) and in vivo (NB cell grafts in NOD-SCID mice) models of NB. NB cell treatment with fendiline induced the expression of the ncRNA NDM29, leading to cell differentiation and to the reduction of the expression of MDRs/ABC transporters linked to multidrug resistance. These events were correlated to higher NB cell susceptibility to cisplatin and, consequently, increased its cytotoxic potency. In vivo, this drug interaction causes an enhanced ability of cisplatin to induce apoptosis in NB masses, resulting in tumor growth reduction and prolonged animal survival rate. Thus, the administration of fendiline might be a possible novel therapeutic approach to increase cisplatin efficacy in aggressive and poorly responsive NB cases.

Identification and dynamic changes of RNAs isolated from RALY-containing ribonucleoprotein complexes.

RALY is a member of the heterogeneous nuclear ribonucleoprotein family (hnRNP), a large family of RNA-binding proteins involved in many aspects of RNA metabolism. Although RALY interactome has been recently characterized, a comprehensive global analysis of RALY-associated RNAs is lacking and the biological function of RALY remains elusive. Here, we performed RIP-seq analysis to identify RALY interacting RNAs and assessed the role of RALY in gene expression. We demonstrate that RALY binds specific coding and non-coding RNAs and associates with translating mRNAs of mammalian cells. Among the identified transcripts, we focused on ANXA1 and H1FX mRNAs, encoding for Annexin A1 and for the linker variant of the histone H1X, respectively. Both proteins are differentially expressed by proliferating cells and are considered as markers for tumorigenesis. We demonstrate that cells lacking RALY expression exhibit changes in the levels of H1FX and ANXA1 mRNAs and proteins in an opposite manner. We also provide evidence for a direct binding of RALY to the U-rich elements present within the 3΄UTR of both transcripts. Thus, our results identify RALY as a poly-U binding protein and as a regulator of H1FX and ANXA1 in mammalian cells.

Regulation of HuR structure and function by dihydrotanshinone-I.

The Human antigen R protein (HuR) is an RNA-binding protein that recognizes U/AU-rich elements in diverse RNAs through two RNA-recognition motifs, RRM1 and RRM2, and post-transcriptionally regulates the fate of target RNAs. The natural product dihydrotanshinone-I (DHTS) prevents the association of HuR and target RNAs in vitro and in cultured cells by interfering with the binding of HuR to RNA. Here, we report the structural determinants of the interaction between DHTS and HuR and the impact of DHTS on HuR binding to target mRNAs transcriptome-wide. NMR titration and Molecular Dynamics simulation identified the residues within RRM1 and RRM2 responsible for the interaction between DHTS and HuR. RNA Electromobility Shifts and Alpha Screen Assays showed that DHTS interacts with HuR through the same binding regions as target RNAs, stabilizing HuR in a locked conformation that hampers RNA binding competitively. HuR ribonucleoprotein immunoprecipitation followed by microarray (RIP-chip) analysis showed that DHTS treatment of HeLa cells paradoxically enriched HuR binding to mRNAs with longer 3'UTR and with higher density of U/AU-rich elements, suggesting that DHTS inhibits the association of HuR to weaker target mRNAs. In vivo, DHTS potently inhibited xenograft tumor growth in a HuR-dependent model without systemic toxicity.

Autophagy inhibition improves the cytotoxic effects of receptor tyrosine kinase inhibitors.

BACKGROUND: A growing field of evidence suggests the involvement of oncogenic receptor tyrosine kinases (RTKs) in cell transformation. Deregulated activity of RTKs in tumors can determine disease progression and therapeutic responses in several types of cancer, including neuroblastoma (NB). Therefore, RTKs targeting is a worthwhile challenge for the oncologists. Nevertheless, acquired resistance to RTK inhibitors (RTKi) remains a serious problem. Autophagy activation is among the possible obstacles for good efficacy of the therapy with RTKi. METHODS: Under different treatment conditions we measured autophagic flux using immunoblot and immunofluorescence assays. Death induction was validated by trypan blue exclusion assay and FACS analysis (calcein-AM/propidium iodide). The NB cell lines SH-SY5Y and Kelly were used for the in vitro study. RESULTS: In order to define whether autophagy might be a limiting factor for the efficacy of RTKi in NB cells, we firstly checked its activation following the treatment with several RTKi. Next, we investigated the possibility to increase their therapeutic efficiency by combining RTKi with autophagy blocking agents in vitro. We exploited the effectiveness of three RTKi either alone or in combination with autophagy inhibitors (Chloroquine-CQ and Spautin-1). We demonstrated that autophagy induction was drug-dependent, and that its inhibition increased the anti-tumor activity of a single RTKi unevenly. We observed that the combined use of blocking agents which impair late autophagy events, such as CQ, and RTKi can be more effective with respect to the use of RTKi alone. CONCLUSIONS: In the present report, we assessed the conditions under which autophagy is activated during the use of different RTKi currently in the pre-clinical evaluation for NB. We summarized the achievements of combined RTK/autophagy inhibitors treatment as a promising approach to enhance the efficacy of RTKi in impairing tumor cells viability.

Positioning Europe for the EPITRANSCRIPTOMICS challenge.

The genetic alphabet consists of the four letters: C, A, G, and T in DNA and C,A,G, and U in RNA. Triplets of these four letters jointly encode 20 different amino acids out of which proteins of all organisms are built. This system is universal and is found in all kingdoms of life. However, bases in DNA and RNA can be chemically modified. In DNA, around 10 different modifications are known, and those have been studied intensively over the past 20 years. Scientific studies on DNA modifications and proteins that recognize them gave rise to the large field of epigenetic and epigenomic research. The outcome of this intense research field is the discovery that development, ageing, and stem-cell dependent regeneration but also several diseases including cancer are largely controlled by the epigenetic state of cells. Consequently, this research has already led to the first FDA approved drugs that exploit the gained knowledge to combat disease. In recent years, the ~150 modifications found in RNA have come to the focus of intense research. Here we provide a perspective on necessary and expected developments in the fast expanding area of RNA modifications, termed epitranscriptomics.

DynaMIT: the dynamic motif integration toolkit.

De-novo motif search is a frequently applied bioinformatics procedure to identify and prioritize recurrent elements in sequences sets for biological investigation, such as the ones derived from high-throughput differential expression experiments. Several algorithms have been developed to perform motif search, employing widely different approaches and often giving divergent results. In order to maximize the power of these investigations and ultimately be able to draft solid biological hypotheses, there is the need for applying multiple tools on the same sequences and merge the obtained results. However, motif reporting formats and statistical evaluation methods currently make such an integration task difficult to perform and mostly restricted to specific scenarios. We thus introduce here the Dynamic Motif Integration Toolkit (DynaMIT), an extremely flexible platform allowing to identify motifs employing multiple algorithms, integrate them by means of a user-selected strategy and visualize results in several ways; furthermore, the platform is user-extendible in all its aspects. DynaMIT is freely available at http://cibioltg.bitbucket.org.

Molecular portraits: the evolution of the concept of transcriptome-based cancer signatures.

Cancer results from dysregulation of multiple steps of gene expression programs. We review how transcriptome profiling has been widely explored for cancer classification and biomarker discovery but resulted in limited clinical impact. Therefore, we discuss alternative and complementary omics approaches.