Circular RNAs arising from synaptic host genes during human neuronal differentiation are modulated by SFPQ RNA-binding protein.

BACKGROUND: Circular RNA (circRNA) molecules, generated through non-canonical back-splicing of exon-exon junctions, have recently been implicated in diverse biological functions including transcriptional regulation and modulation of protein interactions. CircRNAs are emerging as a key component of the complex neural transcriptome implicated in brain development. However, the specific expression patterns and functions of circRNAs in human neuronal differentiation have not been explored. RESULTS: Using total RNA sequencing analysis, we identified expressed circRNAs during the differentiation of human neuroepithelial stem (NES) cells into developing neurons and discovered that many circRNAs originated from host genes associated with synaptic function. Interestingly, when assessing population data, exons giving rise to circRNAs in our dataset had a higher frequency of genetic variants. Additionally, screening for RNA-binding protein sites identified enrichment of Splicing Factor Proline and Glutamine Rich (SFPQ) motifs in increased circRNAs, several of which were reduced by SFPQ knockdown and enriched in SFPQ ribonucleoprotein complexes. CONCLUSIONS: Our study provides an in-depth characterisation of circRNAs in a human neuronal differentiation model and highlights SFPQ as both a regulator and binding partner of circRNAs elevated during neuronal maturation.

RNentropy: an entropy-based tool for the detection of significant variation of gene expression across multiple RNA-Seq experiments.

RNA sequencing (RNA-Seq) has become the experimental standard in transcriptome studies. While most of the bioinformatic pipelines for the analysis of RNA-Seq data and the identification of significant changes in transcript abundance are based on the comparison of two conditions, it is common practice to perform several experiments in parallel (e.g. from different individuals, developmental stages, tissues), for the identification of genes showing a significant variation of expression across all the conditions studied. In this work we present RNentropy, a methodology based on information theory devised for this task, which given expression estimates from any number of RNA-Seq samples and conditions identifies genes or transcripts with a significant variation of expression across all the conditions studied, together with the samples in which they are over- or under-expressed. To show the capabilities offered by our methodology, we applied it to different RNA-Seq datasets: 48 biological replicates of two different yeast conditions; samples extracted from six human tissues of three individuals; seven different mouse brain cell types; human liver samples from six individuals. Results, and their comparison to different state of the art bioinformatic methods, show that RNentropy can provide a quick and in depth analysis of significant changes in gene expression profiles over any number of conditions.

TRIM8 restores p53 tumour suppressor function by blunting N-MYC activity in chemo-resistant tumours.

BACKGROUND: TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway. METHODS: We used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays. RESULTS: We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8. CONCLUSIONS: In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.

Screening autism-associated environmental factors in differentiating human neural progenitors with fractional factorial design-based transcriptomics.

Research continues to identify genetic variation, environmental exposures, and their mixtures underlying different diseases and conditions. There is a need for screening methods to understand the molecular outcomes of such factors. Here, we investigate a highly efficient and multiplexable, fractional factorial experimental design (FFED) to study six environmental factors (lead, valproic acid, bisphenol A, ethanol, fluoxetine hydrochloride and zinc deficiency) and four human induced pluripotent stem cell line derived differentiating human neural progenitors. We showcase the FFED coupled with RNA-sequencing to identify the effects of low-grade exposures to these environmental factors and analyse the results in the context of autism spectrum disorder (ASD). We performed this after 5-day exposures on differentiating human neural progenitors accompanied by a layered analytical approach and detected several convergent and divergent, gene and pathway level responses. We revealed significant upregulation of pathways related to synaptic function and lipid metabolism following lead and fluoxetine exposure, respectively. Moreover, fluoxetine exposure elevated several fatty acids when validated using mass spectrometry-based metabolomics. Our study demonstrates that the FFED can be used for multiplexed transcriptomic analyses to detect relevant pathway-level changes in human neural development caused by low-grade environmental risk factors. Future studies will require multiple cell lines with different genetic backgrounds for characterising the effects of environmental exposures in ASD.

Deficiency of the Heterogeneous Nuclear Ribonucleoprotein U locus leads to delayed hindbrain neurogenesis.

Genetic variants affecting Heterogeneous Nuclear Ribonucleoprotein U (HNRNPU) have been identified in several neurodevelopmental disorders (NDDs). HNRNPU is widely expressed in the human brain and shows the highest postnatal expression in the cerebellum. Recent studies have investigated the role of HNRNPU in cerebral cortical development, but the effects of HNRNPU deficiency on cerebellar development remain unknown. Here, we describe the molecular and cellular outcomes of HNRNPU locus deficiency during in vitro neural differentiation of patient-derived and isogenic neuroepithelial stem cells with a hindbrain profile. We demonstrate that HNRNPU deficiency leads to chromatin remodeling of A/B compartments, and transcriptional rewiring, partly by impacting exon inclusion during mRNA processing. Genomic regions affected by the chromatin restructuring and host genes of exon usage differences show a strong enrichment for genes implicated in epilepsies, intellectual disability, and autism. Lastly, we show that at the cellular level HNRNPU downregulation leads to an increased fraction of neural progenitors in the maturing neuronal population. We conclude that the HNRNPU locus is involved in delayed commitment of neural progenitors to differentiate in cell types with hindbrain profile.

Platelets from patients with visceral obesity promote colon cancer growth.

Several studies highlighted the importance of platelets in the tumor microenvironment due to their ability to interact with other cell types such as leukocytes, endothelial, stromal and cancer cells. Platelets can influence tumor development and metastasis formation through several processes consisting of the secretion of growth factors and cytokines and/or via direct interaction with cancer cells and endothelium. Patients with visceral obesity (VO) are susceptible to pro-thrombotic and pro-inflammatory states and to development of cancer, especially colon cancer. These findings provide us with the impetus to analyze the role of platelets isolated from VO patients in tumor growth and progression with the aim to explore a possible link between platelet activation, obesity and colon cancer. Here, using xenograft colon cancer models, we prove that platelets from patients with visceral obesity are able to strongly promote colon cancer growth. Then, sequencing platelet miRNome, we identify miR-19a as the highest expressed miRNA in obese subjects and prove that miR-19a is induced in colon cancer. Last, administration of miR-19a per se in the xenograft colon cancer model is able to promote colon cancer growth. We thus elect platelets with their specific miRNA abundance as important factors in the tumor promoting microenvironment of patients with visceral obesity.

Presynaptic dysfunction in CASK-related neurodevelopmental disorders.

CASK-related disorders are genetically defined neurodevelopmental syndromes. There is limited information about the effects of CASK mutations in human neurons. Therefore, we sought to delineate CASK-mutation consequences and neuronal effects using induced pluripotent stem cell-derived neurons from two mutation carriers. One male case with autism spectrum disorder carried a novel splice-site mutation and a female case with intellectual disability carried an intragenic tandem duplication. We show reduction of CASK protein in maturing neurons from the mutation carriers, which leads to significant downregulation of genes involved in presynaptic development and of CASK protein interactors. Furthermore, CASK-deficient neurons showed decreased inhibitory presynapse size as indicated by VGAT staining, which may alter the excitatory-inhibitory (E/I) balance in developing neural circuitries. Using in vivo magnetic resonance spectroscopy quantification of GABA in the male mutation carrier, we further highlight the possibility to validate in vitro cellular data in the brain. Our data show that future pharmacological and clinical studies on targeting presynapses and E/I imbalance could lead to specific treatments for CASK-related disorders.

TRIM8 Blunts the Pro-proliferative Action of ΔNp63α in a p53 Wild-Type Background.

The p53 gene family network plays a pivotal role in the control of many biological processes and therefore the right balance between the pro-apoptotic and pro-survival isoforms is key to maintain cellular homeostasis. The stability of the p53 tumor suppressor protein and that of oncogenic ΔNp63α, is crucial to control cell proliferation. The aberrant expression of p53 tumor suppressor protein and oncogenic ΔNp63α contributes to tumorigenesis and significantly affects anticancer drug response. Recently, we demonstrated that TRIM8 increases p53 stability, potentiating its tumor suppressor activity. In this paper, we show that TRIM8 simultaneously reduces the level of the pro-proliferative ΔNp63α protein, in both a proteasomal and caspase-1 dependent way, thereby playing a critical role in the cellular response to DNA damaging agents. Moreover, we provided evidence that ΔNp63α in turn, suppresses TRIM8 gene expression by preventing p53-mediated transactivation of TRIM8, therefore suggesting the existence of a negative feedback loop. These findings indicate that TRIM8 exerts its anticancer power through a joint action that provides on one hand, the activation of the p53 tumor suppressor role, and on the other the quenching of the oncogenic ΔNp63α protein activity. The enhancement of TRIM8 activity may offer therapeutic benefits and improve the management of chemoresistant tumors.

PGC1α: Friend or Foe in Cancer?

The PGC1 family (Peroxisome proliferator-activated receptor γ (PPARγ) coactivators) of transcriptional coactivators are considered master regulators of mitochondrial biogenesis and function. The PGC1α isoform is expressed especially in metabolically active tissues, such as the liver, kidneys and brain, and responds to energy-demanding situations. Given the altered and highly adaptable metabolism of tumor cells, it is of interest to investigate PGC1α in cancer. Both high and low levels of PGC1α expression have been reported to be associated with cancer and worse prognosis, and PGC1α has been attributed with oncogenic as well as tumor suppressive features. Early in carcinogenesis PGC1α may be downregulated due to a protective anticancer role, and low levels likely reflect a glycolytic phenotype. We suggest mechanisms of PGC1α downregulation and how these might be connected to the increased cancer risk that obesity is now known to entail. Later in tumor progression PGC1α is often upregulated and is reported to contribute to increased lipid and fatty acid metabolism and/or a tumor cell phenotype with an overall metabolic plasticity that likely supports drug resistance as well as metastasis. We conclude that in cancer PGC1α is neither friend nor foe, but rather the obedient servant reacting to metabolic and environmental cues to benefit the tumor cell.

TRIM8: Making the Right Decision between the Oncogene and Tumour Suppressor Role.

The TRIM8/GERP protein is a member of the TRIM family defined by the presence of a common domain structure composed of a tripartite motif including a RING-finger, one or two B-box domains, and a coiled-coil motif. The TRIM8 gene maps on chromosome 10 within a region frequently found deleted and rearranged in tumours and transcribes a 3.0-kB mRNA. Its expression is mostly ubiquitously in murine and human tissues, and in epithelial and lymphoid cells, it can be induced by IFNγ. The protein spans 551 aa and is highly conserved during evolution. TRIM8 plays divergent roles in many biological processes, including important functions in inflammation and cancer through regulating various signalling pathways. In regulating cell growth, TRIM8 exerts either a tumour suppressor action, playing a prominent role in regulating p53 tumour suppressor activity, or an oncogene function, through the positive regulation of the NF-κB pathway. The molecular mechanisms underlying this dual role in human cancer will be discussed in depth in this review, and it will highlight the challenge and importance of developing novel therapeutic strategies specifically aimed at blocking the pro-oncogenic arm of the TRIM8 signalling pathway without affecting its tumour suppressive effects.

Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in ALS.

ALS is a devastating and debilitating human disease characterized by the progressive death of upper and lower motor neurons. Although much effort has been made to elucidate molecular determinants underlying the onset and progression of the disorder, the causes of ALS remain largely unknown. In the present work, we have deeply sequenced whole transcriptome from spinal cord ventral horns of post-mortem ALS human donors affected by the sporadic form of the disease (which comprises ~90% of the cases but which is less investigated than the inherited form of the disease). We observe 1160 deregulated genes including 18 miRNAs and show that down regulated genes are mainly of neuronal derivation while up regulated genes have glial origin and tend to be involved in neuroinflammation or cell death. Remarkably, we find strong deregulation of SNAP25 and STX1B at both mRNA and protein levels suggesting impaired synaptic function through SNAP25 reduction as a possible cause of calcium elevation and glutamate excitotoxicity. We also note aberrant alternative splicing but not disrupted RNA editing.

Profiling RNA editing in human tissues: towards the inosinome Atlas.

Adenine to Inosine RNA editing is a widespread co- and post-transcriptional mechanism mediated by ADAR enzymes acting on double stranded RNA. It has a plethora of biological effects, appears to be particularly pervasive in humans with respect to other mammals, and is implicated in a number of diverse human pathologies. Here we present the first human inosinome atlas comprising 3,041,422 A-to-I events identified in six tissues from three healthy individuals. Matched directional total-RNA-Seq and whole genome sequence datasets were generated and analysed within a dedicated computational framework, also capable of detecting hyper-edited reads. Inosinome profiles are tissue specific and edited gene sets consistently show enrichment of genes involved in neurological disorders and cancer. Overall frequency of editing also varies, but is strongly correlated with ADAR expression levels. The inosinome database is available at: http://srv00.ibbe.cnr.it/editing/.

The p53 family member p73 modulates the proproliferative role of IGFBP3 in short children born small for gestational age.

The regulation of insulin-like growth factor-binding protein 3 (IGFBP3) gene expression is complex, because it can be induced by agents that both stimulate and inhibit the proliferation. The principal aim of this study was to investigate whether p73, a member of the p53 gene family, has a role in the regulation of the IGFBP3 expression and whether this regulation occurs in a context of cell survival or death. We demonstrate that IGFBP3 is a direct TAp73α (the p73 isoform that contains the trans-activation domain) target gene and activates the expression of IGFBP3 in actively proliferating cells. As IGFBP3 plays a key role in regulating the growth hormone/insulin-like growth factor type 1 (GH/IGF1) axis, whose alterations in gene expression appear to have a role in the growth failure of children born small for gestational age (SGA), we measured the mRNA expression levels of p73 and IGFBP3 in a group of SGA children. We found that mRNA expression levels of p73 and IGFBP3 are significantly lower in SGA children compared with controls and, in particular, p73 mRNA expression is significantly lower in SGA children with respect to height. Our results shed light on the intricate GH/IGF pathway, suggesting p73 as a good biomarker of the clinical risk for SGA children to remain short in adulthood.

Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity.

Eukaryotic cells contain a population of mitochondria, variable in number and shape, which in turn contain multiple copies of a tiny compact genome (mtDNA) whose expression and function is strictly coordinated with the nuclear one. mtDNA copy number varies between different cell or tissues types, both in response to overall metabolic and bioenergetics demands and as a consequence or cause of specific pathological conditions. Here we present a novel and reliable methodology to assess the effective mtDNA copy number per diploid genome by investigating off-target reads obtained by whole-exome sequencing (WES) experiments. We also investigate whether and how mtDNA copy number correlates with mitochondrial mass, respiratory activity and expression levels. Analyzing six different tissues from three age- and sex-matched human individuals, we found a highly significant linear correlation between mtDNA copy number estimated by qPCR and the frequency of mtDNA off target WES reads. Furthermore, mtDNA copy number showed highly significant correlation with mitochondrial gene expression levels as measured by RNA-Seq as well as with mitochondrial mass and respiratory activity. Our methodology makes thus feasible, at a large scale, the investigation of mtDNA copy number in diverse cell-types, tissues and pathological conditions or in response to specific treatments.

TRIM8 anti-proliferative action against chemo-resistant renal cell carcinoma.

In some tumours, despite a wild-type p53 gene, the p53 pathway is inactivated by alterations in its regulators or by unknown mechanisms, leading to resistance to cytotoxic therapies. Understanding the mechanisms of functional inactivation of wild-type p53 in these tumours may help to define prospective targets for treating cancer by restoring p53 activity. Recently, we identified TRIM8 as a new p53 modulator, which stabilizes p53 impairing its association with MDM2 and inducing the reduction of cell proliferation. In this paper we demonstrated that TRIM8 deficit dramatically impairs p53-mediated cellular responses to chemotherapeutic drugs and that TRIM8 is down regulated in patients affected by clear cell Renal Cell Carcinoma (ccRCC), an aggressive drug-resistant cancer showing wild-type p53. These results suggest that down regulation of TRIM8 might be an alternative way to suppress p53 activity in RCC. Interestingly, we show that TRIM8 expression recovery in RCC cell lines renders these cells sensitive to chemotherapeutic treatments following p53 pathway re-activation. These findings provide the first mechanistic link between TRIM8 and the drug resistance of ccRCC and suggest more generally that TRIM8 could be used as enhancer of the chemotherapy efficacy in cancers where p53 is wild-type and its pathway is defective.