Stefano de Pretis's research while affiliated with San Raffaele Scientific Institute and other places

Publications (27)

Article
The nervous system requires metabolites and oxygen supplied by the neurovascular network, but this necessitates close apposition of neurons and endothelial cells. We find motor neurons attract vessels with long-range VEGF signaling, but endothelial cells in the axonal pathway are an obstacle for establishing connections with muscles. It is unclear...
Article
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Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosine kinase BCR-ABL1 fusion protein, which deregulate transcription and mRNA translation. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge to cure CML patients. Here, we reveal that the m6A m...
Chapter
The field of transcriptional regulation generally assumes that changes in transcripts levels reflect changes in transcriptional status of the corresponding gene. While this assumption might hold true for a large population of transcripts, a considerable and still unrecognized fraction of the variation might involve other steps of the RNA lifecycle,...
Article
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Despite gene expression programs being notoriously complex, RNA abundance is usually assumed as a proxy for transcriptional activity. Recently developed approaches, able to disentangle transcriptional and post-transcriptional regulatory processes, have revealed a more complex scenario. It is now possible to work out how synthesis, processing and de...
Article
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Post-transcriptional repression of gene expression by miRNAs occurs through transcript destabilization or translation inhibition. mRNA decay is known to account for most miRNA-dependent repression. However, because transcript decay occurs co-translationally, whether target translation is a requirement for miRNA-dependent transcript destabilization...
Article
Full-text available
The quantification of the kinetic rates of RNA synthesis, processing, and degradation are largely based on the integrative analysis of total and nascent transcription, the latter being quantified through RNA metabolic labeling. We developed INSPEcT-, a computational method based on the mathematical modeling of premature and mature RNA expression th...
Article
Full-text available
The abundance of RNA species and their response to perturbations are set by the kinetics rates of RNA synthesis, processing, and degradation. However, the visualization, interpretation, and manipulation of these data require familiarity with mathematical modeling and command line tools. INSPEcT-GUI is an R-Shiny interface that allows researchers wi...
Article
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It has been known for a few decades that transcripts can be marked by dozens of different modifications. Yet, we are just at the beginning of charting these marks and understanding their functional impact. High-quality methods were developed for the profiling of some of these marks, and approaches to finely study their impact on specific phases of...
Preprint
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Posttranscriptional repression by microRNA (miRNA) occurs through transcript destabilization or translation inhibition. Whereas RNA degradation explains most miRNA-dependent repression, transcript decay occurs co-translationally, raising questions regarding the requirement of target translation to miRNA-dependent transcript destabilization. To asse...
Article
Upon activation, lymphocytes exit quiescence and undergo substantial increases in cell size, accompanied by activation of energy-producing and anabolic pathways, widespread chromatin decompaction, and elevated transcriptional activity. These changes depend upon prior induction of the Myc transcription factor, but how Myc controls them remains uncle...
Preprint
Upon activation, lymphocytes exit quiescence and undergo substantial increases in cell size, accompanied by activation of energy-producing and anabolic pathways, widespread chromatin decompaction and elevated transcriptional activity. These changes depend upon prior induction of the Myc transcription factor, but how Myc controls them remains unclea...
Preprint
Full-text available
The kinetic rates of RNA synthesis, processing and degradation determine the dynamics of transcriptional regulation by governing both the abundance and the responsiveness to modulations of premature and mature RNA species. The study of RNA dynamics is largely based on the integrative analysis of total and nascent transcription, with the latter bein...
Article
Full-text available
N6-methyladenosine (m6A) is the most abundant RNA modification. It has been involved in the regulation of RNA metabolism, including degradation and translation, in both physiological and disease conditions. A recent study showed that m6A-mediated degradation of key transcripts also plays a role in the control of T cells homeostasis and IL-7 induced...
Article
Overexpression of the MYC transcription factor causes its widespread interaction with regulatory elements in the genome but leads to the up- and down-regulation of discrete sets of genes. The molecular determinants of these selective transcriptional responses remain elusive. Here, we present an integrated time-course analysis of transcription and m...
Article
Pervasive transcription of the human genome results in a heterogeneous mix of coding RNAs and long noncoding RNAs (lncRNAs). Only a small fraction of lncRNAs have demonstrated regulatory functions, thus making functional lncRNAs difficult to distinguish from nonfunctional transcriptional byproducts. This difficulty has resulted in numerous competin...
Preprint
The pervasive transcription of the human genome results in a heterogeneous mix of coding and long non-coding RNAs (lncRNAs). Only a small fraction of lncRNAs possess demonstrated regulatory functions, making it difficult to distinguish functional lncRNAs from non-functional transcriptional byproducts. This has resulted in numerous competing classif...
Article
Full-text available
Next-generation sequencing (NGS) technologies have deeply changed our understanding of cellular processes by delivering an astonishing amount of data at affordable prices; nowadays, many biology laboratories have already accumulated a large number of sequenced samples. However, managing and analyzing these data poses new challenges, which may easil...
Article
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The increasing availability of resequencing data has led to a better understanding of the most important genes in cancer development. Nevertheless, the mutational landscape of many tumor types is heterogeneous and encompasses a long tail of potential driver genes that are systematically excluded by currently available methods due to the low frequen...
Article
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The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA deca...
Article
Myc binds DNA with a preference for the E-box consensus CACGTG. In vivo, however, DNA recognition is primarily determined by chromatin context, preceding sequence-specific DNA binding. Myc preferentially associates with active/poised promoters and, to a lesser extent, distal enhancer elements. When expressed at high levels, Myc targets virtually al...
Article
Upon recruitment to active enhancers and promoters, RNA polymerase II (Pol II) generates short non-coding transcripts of unclear function. The mechanisms that control the length and the amount of ncRNAs generated by cis-regulatory elements are largely unknown. Here, we show that the adaptor protein WDR82 and its associated complexes actively limit...
Article
Full-text available
Background Numerous methods are available to profile several epigenetic marks, providing data with different genome coverage and resolution. Large epigenomic datasets are then generated, and often combined with other high-throughput data, including RNA-seq, ChIP-seq for transcription factors (TFs) binding and DNase-seq experiments. Despite the nume...
Article
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The gene expression programs regulated by the Myc transcription factor were evaluated by integrated genome-wide profiling of Myc binding sites, chromatin marks and RNA expression in several biological models. Our results indicate that Myc directly drives selective transcriptional regulation, which in certain physiological conditions may indirectly...
Article
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Cellular mRNA levels originate from the combined action of multiple regulatory processes, which can be recapitulated by the rates of pre-mRNA synthesis, pre-mRNA processing, and mRNA degradation. Recent experimental and computational advances set the basis to study these intertwined levels of regulation. Nevertheless, software for the comprehensive...
Article
Full-text available
A multi-layered set of epigenetic marks, including post-translational modifications of histones and methylation of DNA, is finely tuned to define the epigenetic state of chromatin in any given cell type under specific conditions. Recently, the knowledge about the combinations of epigenetic marks occurring in the genome of different cell types under...
Article
Full-text available
The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci 1 . Recent work suggested that rather than up-and downregulating selected groups of genes 1–3 , Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription 4,5 . How...
Article
Full-text available
A key challenge in the analysis of cancer genomes is the identification of driver genes from the vast number of mutations present in a cohort of patients. DOTS-Finder is a new tool that allows the detection of driver genes through the sequential application of functional and frequentist approaches, and is specifically tailored to the analysis of fe...

Citations

... Furthermore, his group elucidated the consequences of METTL3 altered subcellular localization, showing that cytoplasmic METTL3 has a role independent from its nuclear catalytic activity. In fact, the knockdown of METTL3 in the cytoplasm affects translation in CML and he hypothesized that the translation-promoting effect on methylated mRNAs could represent a general mechanism for cells where METTL3 is localized in the cytoplasm (Ianniello et al., 2021). Moreover, Fatica demonstrated the importance of using m6A effector inhibitors that could represent an effective therapy for CML in the future. ...
... Enriching transcripts associated with chromatin or active RNA polymerase II accurately identified the de novo synthesized RNAs and revealed new features of gene expression (Core et al., 2008;Kwak et al., 2013;Mayer and Churchman, 2016), but with little quantitative information. In fact, these approaches cannot disentangle the presence of transcripts or the velocity of RNA polymerase II from the efficiency of RNA synthesis, lacking the information to measure the transcriptional output (Furlan et al., 2020a). These protocols could also be influenced by binding of non-specific RNAs or proteins. ...
... Introduction 1 understanding the function of miRNAs. Although much progress has been made, target 6 identification remains a challenge because of the limited understanding of the molecular 7 basis of miRNA-target coupling, but also due to the context-dependence of 8 post-transcriptional regulation and miRNA mode of action [4]. Generally, miRNAs 9 downregulate proteins through a combination of translational inhibition and promotion 10 of mRNA decay [5][6][7], even though which mechanisms of action of microRNAs are the 11 most dominant remains a matter of debate. ...
... All the described approaches are relatively laborious and require biochemical separation, thus being very sensitive to the accuracy of labelled molecules enrichment. They also require relatively high amount of starting material and might contain up to 30% unlabelled RNA contaminants in the labelled fraction (Furlan et al., 2020b). To circumvent these limitations, new metabolic labelling protocols were recently developed. ...
... Furthermore, computer algorithms based on RNA-Seq are still under continuous development. For example, INSPEcT [97] was recently designed to calculate RNA kinetic rates based on time course RNA-seq data, or to estimate stability by calculating the difference between premature and mature RNA expression [100]. Going forward, stQTLs identified with more accurate mRNA stability profile estimations may further our understanding of how genetic variants regulate gene expression. ...
... Miscall analysis is currently limited based on the constraints of the base caller and the quality of the alignments. 4,28,29 Nanopore Detection of off-Target Biological tRNAs. We aligned nanopore reads for each of the purified biological tRNA samples against the total E. coli tRNA sequence reference (Table S3, Figure S8). ...
... Our findings further support the idea that the transition to the tumor tissue state involves specific epigenetic and transcriptional profile changes, which are dictated by both MYC overexpression and the initial tissue state. In part, this could be a direct consequence of MYC's regulation of epigenetic programs, whether through miR-17-92 which controls the expression of certain chromatin-modifying genes [57], physical interaction with chromatin-remodeling complexes [58][59][60], direct regulation of DNA methyltransferases [61], or other mechanisms of regulation of chromatin regulatory genes [57,[62][63][64]. Since tumorigenesis is a multi-step process [65], it is probable that a step-wise succession of feedback loops involving gene expression changes gives rise to new cellular states. ...
... This model is implemented, with various assumptions, by different tools [cDTA (Sun et al., 2012), DRiLL (Rabani et al., 2014), INSPEcT (de Pretis et al., 2015) and pulseR (Uvarovskii and Dieterich, 2017)], which rely on the quantification of both nascent and total RNA species, the former profiled through RNA metabolic labeling (Dolken et al., 2008). Recently, novel approaches are being developed that do not require the quantification of nascent RNA, to estimate the full set (Furlan et al., 2019), or a subset of the kinetic rates (Zeisel et al., 2011;Gray et al., 2014;La Manno et al., 2018). Despite the availability of these tools, anticipating the outcome of the joint contribution of various RNA life-cycle stages can be far from trivial. ...
... Building up on the previous study, researchers quantified RNA dynamics in T cells, using bioinformatic analysis, to reveal how transcripts are regulated by m 6 A. In the context of T cell homeostasis, m 6 A depletion is reported to globally slow down the rates of all stages of the RNA life cycle by delaying RNA synthesis rates, impairing RNA processing rates and hindering SOCS mRNA decay rates. All these effects may directly or indirectly upset T cell differentiation [64]. Interestingly, these findings suggest that T cell-targeted delivery of m 6 A modifying agents could be an eminent step in cancer immunotherapy [31,63,65]. ...
... We next compared the RNAPII PI of the three sets of target genes in ESCs ( Figure 4D). We found genes bound specifically by MYC, a factor well-documented for its role in RNAPII pause release (Baluapuri et al., Lu et al., 2015;De Pretis et al., 2017;Price, 2010;Rahl et al., 2010), had a similar level of RNAPII pausing compared to those bound specifically by LSD1 ( Figure 4D), and consistent with our observation that the co-occupied sites are enriched for RNAPII pausing factors, we found that genes co-occupied by LSD1 and MYC exhibited significantly higher RNAPII pausing than those with LSD1 or MYC alone. iScience Article MYC binding at co-localized target genes is dependent on the presence of LSD1 ...