Multiscale modeling uncovers 7q11.23 copy number variation-dependent changes in ribosomal biogenesis and neuronal maturation and excitability.

Copy number variation (CNV) at 7q11.23 causes Williams-Beuren syndrome (WBS) and 7q microduplication syndrome (7Dup), neurodevelopmental disorders (NDDs) featuring intellectual disability accompanied by symmetrically opposite neurocognitive features. Although significant progress has been made in understanding the molecular mechanisms underlying 7q11.23-related pathophysiology, the propagation of CNV dosage across gene expression layers and their interplay remains elusive. Here we uncovered 7q11.23 dosage-dependent symmetrically opposite dynamics in neuronal differentiation and intrinsic excitability. By integrating transcriptomics, translatomics, and proteomics of patient-derived and isogenic induced neurons, we found that genes related to neuronal transmission follow 7q11.23 dosage and are transcriptionally controlled, while translational factors and ribosomal genes are posttranscriptionally buffered. Consistently, we found phosphorylated RPS6 (p-RPS6) downregulated in WBS and upregulated in 7Dup. Surprisingly, p-4EBP was changed in the opposite direction, reflecting dosage-specific changes in total 4EBP levels. This highlights different dosage-sensitive dyregulations of the mTOR pathway as well as distinct roles of p-RPS6 and p-4EBP during neurogenesis. Our work demonstrates the importance of multiscale disease modeling across molecular and functional layers, uncovers the pathophysiological relevance of ribosomal biogenesis in a paradigmatic pair of NDDs, and uncouples the roles of p-RPS6 and p-4EBP as mechanistically actionable relays in NDDs.

miRNA-mediated inhibition of an actomyosin network in hippocampal pyramidal neurons restricts sociability in adult male mice.

Social deficits are frequently observed in patients suffering from neurodevelopmental disorders, but the molecular mechanisms regulating sociability are still poorly understood. We recently reported that the loss of the microRNA (miRNA) cluster miR-379-410 leads to hypersocial behavior and anxiety in mice. Here, we show that ablating miR-379-410 in excitatory neurons of the postnatal mouse hippocampus recapitulates hypersociability, but not anxiety. At the cellular level, miR-379-410 loss in excitatory neurons leads to larger dendritic spines, increased excitatory synaptic transmission, and upregulation of an actomyosin gene network. Re-expression of three cluster miRNAs, as well as pharmacological inhibition of the actomyosin activator ROCK, is sufficient to reinstate normal sociability in miR-379-410 knockout mice. Several actomyosin genes and miR-379-410 family members are reciprocally dysregulated in isogenic human induced pluripotent stem cell (iPSC)-derived neurons harboring a deletion present in patients with Williams-Beuren syndrome, characterized by hypersocial behavior. Together, our results show an miRNA-actomyosin pathway involved in social behavior regulation.

GTF2I dosage regulates neuronal differentiation and social behavior in 7q11.23 neurodevelopmental disorders.

Copy number variations at 7q11.23 cause neurodevelopmental disorders with shared and opposite manifestations. Deletion causes Williams-Beuren syndrome featuring hypersociability, while duplication causes 7q11.23 microduplication syndrome (7Dup), frequently exhibiting autism spectrum disorder (ASD). Converging evidence indicates GTF2I as key mediator of the cognitive-behavioral phenotypes, yet its role in cortical development and behavioral hallmarks remains largely unknown. We integrated proteomic and transcriptomic profiling of patient-derived cortical organoids, including longitudinally at single-cell resolution, to dissect 7q11.23 dosage-dependent and GTF2I-specific disease mechanisms. We observed dosage-dependent impaired dynamics of neural progenitor proliferation, transcriptional imbalances, and highly specific alterations in neuronal output, leading to precocious excitatory neuron production in 7Dup, which was rescued by restoring physiological GTF2I levels. Transgenic mice with Gtf2i duplication recapitulated progenitor proliferation and neuronal differentiation defects alongside ASD-like behaviors. Consistently, inhibition of lysine demethylase 1 (LSD1), a GTF2I effector, was sufficient to rescue ASD-like phenotypes in transgenic mice, establishing GTF2I-LSD1 axis as a molecular pathway amenable to therapeutic intervention in ASD.

Long non-coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation.

Transition from proliferative-to-invasive phenotypes promotes metastasis and therapy resistance in melanoma. Reversion of the invasive phenotype, however, is challenged by the poor understanding of mechanisms underlying its maintenance. Here, we report that the lncRNA TINCR is down-regulated in metastatic melanoma and its silencing increases the expression levels of invasive markers, in vitro migration, in vivo tumor growth, and resistance to BRAF and MEK inhibitors. The critical mediator is ATF4, a central player of the integrated stress response (ISR), which is activated in TINCR-depleted cells in the absence of starvation and eIF2α phosphorylation. TINCR depletion increases global protein synthesis and induces translational reprogramming, leading to increased translation of mRNAs encoding ATF4 and other ISR proteins. Strikingly, re-expression of TINCR in metastatic melanoma suppresses the invasive phenotype, reduces numbers of tumor-initiating cells and metastasis formation, and increases drug sensitivity. Mechanistically, TINCR interacts with mRNAs associated with the invasive phenotype, including ATF4, preventing their binding to ribosomes. Thus, TINCR is a suppressor of the melanoma invasive phenotype, which functions in nutrient-rich conditions by repressing translation of selected ISR RNAs.

High-throughput screening identifies histone deacetylase inhibitors that modulate GTF2I expression in 7q11.23 microduplication autism spectrum disorder patient-derived cortical neurons.

BACKGROUND: Autism spectrum disorder (ASD) is a highly prevalent neurodevelopmental condition affecting almost 1% of children, and represents a major unmet medical need with no effective drug treatment available. Duplication at 7q11.23 (7Dup), encompassing 26-28 genes, is one of the best characterized ASD-causing copy number variations and offers unique translational opportunities, because the hemideletion of the same interval causes Williams-Beuren syndrome (WBS), a condition defined by hypersociability and language strengths, thereby providing a unique reference to validate treatments for the ASD symptoms. In the above-indicated interval at 7q11.23, defined as WBS critical region, several genes, such as GTF2I, BAZ1B, CLIP2 and EIF4H, emerged as critical for their role in the pathogenesis of WBS and 7Dup both from mouse models and human studies. METHODS: We performed a high-throughput screening of 1478 compounds, including central nervous system agents, epigenetic modulators and experimental substances, on patient-derived cortical glutamatergic neurons differentiated from our cohort of induced pluripotent stem cell lines (iPSCs), monitoring the transcriptional modulation of WBS interval genes, with a special focus on GTF2I, in light of its overriding pathogenic role. The hits identified were validated by measuring gene expression by qRT-PCR and the results were confirmed by western blotting. RESULTS: We identified and selected three histone deacetylase inhibitors (HDACi) that decreased the abnormal expression level of GTF2I in 7Dup cortical glutamatergic neurons differentiated from four genetically different iPSC lines. We confirmed this effect also at the protein level. LIMITATIONS: In this study, we did not address the molecular mechanisms whereby HDAC inhibitors act on GTF2I. The lead compounds identified will now need to be advanced to further testing in additional models, including patient-derived brain organoids and mouse models recapitulating the gene imbalances of the 7q11.23 microduplication, in order to validate their efficacy in rescuing phenotypes across multiple functional layers within a translational pipeline towards clinical use. CONCLUSIONS: These results represent a unique opportunity for the development of a specific class of compounds for treating 7Dup and other forms of intellectual disability and autism.

PRMT1-mediated methylation of the microprocessor-associated proteins regulates microRNA biogenesis.

MicroRNA (miRNA) biogenesis is a tightly controlled multi-step process operated in the nucleus by the activity of the Microprocessor and its associated proteins. Through high resolution mass spectrometry (MS)- proteomics we discovered that this complex is extensively methylated, with 84 methylated sites associated to 19 out of its 24 subunits. The majority of the modifications occurs on arginine (R) residues (61), leading to 81 methylation events, while 30 lysine (K)-methylation events occurs on 23 sites of the complex. Interestingly, both depletion and pharmacological inhibition of the Type-I Protein Arginine Methyltransferases (PRMTs) lead to a widespread change in the methylation state of the complex and induce global decrease of miRNA expression, as a consequence of the impairment of the pri-to-pre-miRNA processing step. In particular, we show that the reduced methylation of the Microprocessor subunit ILF3 is linked to its diminished binding to the pri-miRNAs miR-15a/16, miR-17-92, miR-301a and miR-331. Our study uncovers a previously uncharacterized role of R-methylation in the regulation of miRNA biogenesis in mammalian cells.

Mass Spectrometry-Based Proteomics to Unveil the Non-coding RNA World.

The interaction between non-coding RNAs (ncRNAs) and proteins is crucial for the stability, localization and function of the different classes of ncRNAs. Although ncRNAs, when embedded in various ribonucleoprotein (RNP) complexes, control the fundamental processes of gene expression, their biological functions and mechanisms of action are still largely unexplored. Mass Spectrometry (MS)-based proteomics has emerged as powerful tool to study the ncRNA world: on the one hand, by identifying the proteins interacting with distinct ncRNAs; on the other hand, by measuring the impact of ncRNAs on global protein levels. Here, we will first provide a concise overview on the basic principles of MS-based proteomics for systematic protein identification and quantification; then, we will recapitulate the main approaches that have been implemented for the screening of ncRNA interactors and the dissection of ncRNA-protein complex composition. Finally, we will describe examples of various proteomics strategies developed to characterize the effect of ncRNAs on gene expression, with a focus on the systematic identification of microRNA (miRNA) targets.

Spatiotemporal plasticity of miRNAs functions: The miR-17-92 case.

The functional effect of a specific miRNA is tightly linked to the transcriptome, thus having the potential to elicit distinct outcomes in different cellular states. Our recent discovery of a dual role of the miR-17-92 cluster, which shifts from oncogene to tumor suppressor during lymphoma progression, exemplifies the spatiotemporal plasticity of miRNAs.

MS-analysis of SILAC-labeled MYC-driven B lymphoma cells overexpressing miR-17-19b.

Micro RNAs (miRNAs) are small non-coding RNAs, which dampen gene expression by repressing translation and/or inducing degradation of target-mRNAs. Although the role of miR-17-19b (a truncated version of miR-17-92 cluster) is well documented in MYC-driven B cell lymphomagenesis, little is known about the function of the cluster in the maintenance of full-blown lymphomas. We employed SILAC-based quantitative proteomics to identify miR-17-19b targets upon a mild overexpression of the cluster in B cell lymphomas, established from λ-MYC transgenic mice. The proteomics data described in detail in this study, whose follow up analysis with MaxQuant algorithm is part of the recent publication (Mihailovich et al., 2015) [1], are deposited to the ProteomeXchange Consortium via the PRIDE partner repository, with the accession code PRIDE: PXD002810.

miR-17-92 fine-tunes MYC expression and function to ensure optimal B cell lymphoma growth.

The synergism between c-MYC and miR-17-19b, a truncated version of the miR-17-92 cluster, is well-documented during tumor initiation. However, little is known about miR-17-19b function in established cancers. Here we investigate the role of miR-17-19b in c-MYC-driven lymphomas by integrating SILAC-based quantitative proteomics, transcriptomics and 3' untranslated region (UTR) analysis upon miR-17-19b overexpression. We identify over one hundred miR-17-19b targets, of which 40% are co-regulated by c-MYC. Downregulation of a new miR-17/20 target, checkpoint kinase 2 (Chek2), increases the recruitment of HuR to c-MYC transcripts, resulting in the inhibition of c-MYC translation and thus interfering with in vivo tumor growth. Hence, in established lymphomas, miR-17-19b fine-tunes c-MYC activity through a tight control of its function and expression, ultimately ensuring cancer cell homeostasis. Our data highlight the plasticity of miRNA function, reflecting changes in the mRNA landscape and 3' UTR shortening at different stages of tumorigenesis.

ARGONAUTE2 cooperates with SWI/SNF complex to determine nucleosome occupancy at human Transcription Start Sites.

Argonaute (AGO) proteins have a well-established role in post-transcriptional regulation of gene expression as key component of the RNA silencing pathways. Recent evidence involves AGO proteins in mammalian nuclear processes such as transcription and splicing, though the mechanistic aspects of AGO nuclear functions remain largely elusive. Here, by SILAC-based interaction proteomics, we identify the chromatin-remodelling complex SWI/SNF as a novel AGO2 interactor in human cells. Moreover, we show that nuclear AGO2 is loaded with a novel class of Dicer-dependent short RNAs (sRNAs), that we called swiRNAs, which map nearby the Transcription Start Sites (TSSs) bound by SWI/SNF. The knock-down of AGO2 decreases nucleosome occupancy at the first nucleosome located downstream of TSSs in a swiRNA-dependent manner. Our findings indicate that in human cells AGO2 binds SWI/SNF and a novel class of sRNAs to establish nucleosome occupancy on target TSSs.

Widespread generation of alternative UTRs contributes to sex-specific RNA binding by UNR.

Upstream of N-ras (UNR) is a conserved RNA-binding protein that regulates mRNA translation and stability by binding to sites generally located in untranslated regions (UTRs). In Drosophila, sex-specific binding of UNR to msl2 mRNA and the noncoding RNA roX is believed to play key roles in the control of X-chromosome dosage compensation in both sexes. To investigate broader sex-specific functions of UNR, we have identified its RNA targets in adult male and female flies by high-throughput RNA binding and transcriptome analysis. Here we show that UNR binds to a large set of protein-coding transcripts and to a smaller set of noncoding RNAs in a sex-specific fashion. The analyses also reveal a strong correlation between sex-specific binding of UNR and sex-specific differential expression of UTRs in target genes. Validation experiments indicate that UNR indeed recognizes sex-specifically processed transcripts. These results suggest that UNR exploits the transcript diversity generated by alternative processing and alternative promoter usage to bind and regulate target genes in a sex-specific manner.

ß-Secretase activity in rat astrocytes: translational block of BACE1 and modulation of BACE2 expression.

BACE1 and BACE2 are two closely related membrane-bound aspartic proteases. BACE1 is widely recognized as the neuronal ß-secretase that cleaves the amyloid-ß precursor protein, thus allowing the production of amyloid-ß, i.e. the peptide that has been proposed to trigger the neurodegenerative process in Alzheimer's disease. BACE2 has ubiquitous expression and its physiological and pathological role is still unclear. In light of a possible role of glial cells in the accumulation of amyloid-ß in brain, we have investigated the expression of these two enzymes in primary cultures of astrocytes. We show that astrocytes possess ß-secretase activity and produce amyloid-ß because of the activity of BACE2, but not BACE1, the expression of which is blocked at the translational level. Finally, our data demonstrate that changes in the astrocytic phenotype during neuroinflammation can produce both a negative as well as a positive modulation of ß-secretase activity, also depending on the differential responsivity of the brain regions.

Eukaryotic cold shock domain proteins: highly versatile regulators of gene expression.

Cold shock domain (CSD)-containing proteins have been found in all three domains of life and function in a variety of processes that are related, for the most part, to post-transcriptional gene regulation. The CSD is an ancient beta-barrel fold that serves to bind nucleic acids. The CSD is structurally and functionally similar to the S1 domain, a fold with otherwise unrelated primary sequence. The flexibility of the CSD/S1 domain for RNA recognition confers an enormous functional versatility to the proteins that contain them. This review summarizes the current knowledge on eukaryotic CSD/S1 domain-containing proteins with a special emphasis on UNR (upstream of N-ras), a member of this family with multiple copies of the CSD.

Dual sex-specific functions of Drosophila Upstream of N-ras in the control of X chromosome dosage compensation.

Dosage compensation in Drosophila melanogaster involves the assembly of the MSL-2-containing dosage compensation complex (DCC) on the single X chromosome of male flies. Translational repression of msl-2 mRNA blocks this process in females. Previous work indicated that the ubiquitous protein Upstream of N-ras (UNR) is a necessary co-factor for msl-2 repression in vitro. Here, we explore the function of UNR in vivo. Hypomorphic Unr mutant flies showed DCC assembly on high-affinity sites in the female X chromosomes, confirming that UNR inhibits dosage compensation in female flies. Unexpectedly, male mutant flies and UNR-depleted SL2 cells showed decreased DCC binding to the X chromosome, suggesting a role for UNR in DCC assembly or targeting. Consistent with this possibility, UNR overexpression resulted in moderate loss of DCC from the male X chromosome and predominant male lethality. Immunoprecipitation experiments revealed that UNR binds to roX1 and roX2, the non-coding RNA components of the DCC, providing possible targets for UNR function in males. These results uncover dual sex-specific functions of UNR in dosage compensation: to repress DCC formation in female flies and to promote DCC assembly on the male X chromosome.

BACE1 expression and activity: relevance in Alzheimer's disease.

A turning point of research in Alzheimer's disease was undoubtedly the discovery of BACE1, the amyloid-beta precursor protein-cleaving enzyme that initiates the generation of amyloid-beta, the peptide strongly suspected to be responsible for neuronal malfunction and death. Several research groups started a race to identify the best inhibitor of BACE1 activity. On the other hand, basic researchers are evaluating the changes in BACE1 expression and activity with the aim to better understand the pathogenetic process of the disease. Along this second line of research, in the last few years many important results have been reported in various experimental models, as well as in Alzheimer's disease patients. As a consequence, new pathogenetic paradigms have been developed. We have reviewed these reports trying to highlight contrasting viewpoints, data awaiting final confirmation, and promising perspectives.

Complex translational regulation of BACE1 involves upstream AUGs and stimulatory elements within the 5' untranslated region.

BACE1 is the protease responsible for the production of amyloid-beta peptides that accumulate in the brain of Alzheimer's disease (AD) patients. BACE1 expression is regulated at the transcriptional, as well as post-transcriptional level. Very high BACE1 mRNA levels have been observed in pancreas, but the protein and activity were found mainly in brain. An up-regulation of the protein has been described in some AD patients without a change in transcript levels. The features of BACE1 5' untranslated region (5' UTR), such as the length, GC content, evolutionary conservation and presence of upstream AUGs (uAUGs), indicate an important regulatory role of this 5' UTR in translational control. We demonstrate that, in brain and pancreas, almost all of the native BACE1 mRNA contains the full-length 5' UTR. RNA transfection and in vitro translation show that translation is mainly inhibited by the presence of the uAUGs. We provide a mutational analysis that highlight the second uAUG as the main inhibitory element while mutations of all four uAUGs fully de-repress translation. Furthermore, we have evidence that a sequence within the region 222-323 of the BACE1 5' UTR has a stimulatory effect on translation that might depend on the presence of trans-acting factors.

Translational regulation of BACE-1 expression in neuronal and non-neuronal cells.

As the main beta-secretase of the central nervous system, BACE-1 is a key protein in the pathogenesis of Alzheimer's disease. Excessive expression of the protein might cause an overproduction of the neurotoxic beta-amyloid peptide. Therefore, a tight regulation of BACE-1 expression is expected in vivo. In addition to a possible transcriptional control, the BACE-1 transcript leader contains features that might constitute mechanisms of translational regulation of protein expression. Moreover, recent work has revealed an increase of BACE-1 protein and beta-secretase activity in some Alzheimer's disease patients, although a corresponding increase of transcript has not been reported. Here we show that BACE-1 translation could be modulated at multiple stages. The presence of several upstream ATGs strongly reduces the translation of the main open reading frame. This inhibition could be overcome with conditions that favour skipping of upstream ATGs. We also report an alternative splicing of the BACE-1 transcript leader that reduces the number of upstream ATGs. Finally, we show that translation driven by the BACE-1 transcript leader is increased in activated astrocytes independently of the splicing event, indicating yet another mechanism of translational control. Our findings might explain why increases in BACE-1 protein or activity are reported in the brain of Alzheimer's disease patients even in the absence of changes in transcript levels.

Translational control of Scamper expression via a cell-specific internal ribosome entry site.

The mRNA of Scamper, a putative intracellular calcium channel activated by sphingosylphosphocholine, contains a long 5' transcript leader with several upstream AUGs. In this work we have investigated the role this sequence plays in the translational control of Scamper expression. The cytosolic transcription machinery of a T7 RNA polymerase recombinant vaccinia virus was used to avoid artifacts arising from cryptic promoters or mRNA processing. Based on transient transfection experiments of dicistronic and bi-monocistronic plasmids expressing reporter genes, we present evidence that the 5' transcript leader of Scamper contains a functional internal ribosome entry site (IRES). Our data indicate that Scamper translation in Madin-Darby canine kidney cells is driven by a cap-independent mechanism supported by the IRES activity of its mRNA. Finally, the Scamper IRES appears to be the first IRES with specificity for kidney epithelial cells.