Papers by Alessandro Fatica
The METTL3/METTL14 m6A methylation complex plays a crucial role in Chronic Myeloid Leukemia survival by regulating MYC expression
Chronic Myeloid Leukemia (CML) is a malignant myeloproliferative disease caused by a chromosomal ... more Chronic Myeloid Leukemia (CML) is a malignant myeloproliferative disease caused by a chromosomal translocation that produces the constitutively activated tyrosine kinase BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKIs) are the first-choice treatment. However, resistance to TKIs remains a challenge for curing CML patients. To gain insight into the role of m6A modification in CML, we analyze the role of the METTL3/METTL14 m6A writing complex in the BCR-ABL1+ K562 CML cellular model. We show that knockdown of METTL3 and METTL14 strongly impaired proliferation of both TKI-sensitive and TKI-resistant cells. Furthermore, we demonstrate that MYC oncogenes is highly m6A methylated in CML and that m6A marks are required for its efficient expression. Therefore, our findings demonstrate an important role for m6A methylation in CML and show that targeting the METTL3/METTL14 complex may represent a promising therapeutic strategy for TKIs resistant CML cells
A positive feed-forward regulatory loop between METTL3 and WTAP sustains the oncogenic role of the m6A methylation complex in myeloid leukemia
The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumours, including, acut... more The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumours, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N6-Methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Our results indicate the existence of a positive feedforward regulatory loop, where METTL3 upregulates WTAP, which is relevant to increase WTAP expr...
Rrp14p is required for pre-rRNA processing
<b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal ... more <b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal subunit synthesis and for correct positioning of the mitotic spindle during mitosis"Nucleic Acids Research 2007;35(4):1354-1366.Published online 01 Feb 2007PMCID:PMC1849896.© 2007 The Author(s) () Schematic diagram of the pre-rRNA showing the processing sites and locations of oligonucleotide probes used. () Northern analyses of high molecular weight RNA separated on a 1.2% agarose/formaldehyde gel. () Northern analyses of low molecular weight RNA separated on a 6% polyacrylamide/urea gel. RNA species are labeled on the right of the northern panels. Oligonucleotide probes used are on the right.
Depletion of Rrp14p impairs growth and 60S subunit synthesis
<b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal ... more <b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal subunit synthesis and for correct positioning of the mitotic spindle during mitosis"Nucleic Acids Research 2007;35(4):1354-1366.Published online 01 Feb 2007PMCID:PMC1849896.© 2007 The Author(s) () Growth of (crosses) and otherwise isogenic wild-type (open boxes) strains following transfer to glucose medium. () Western analysis of the depletion of HA–Rrp14p on glucose medium. ( and ) Pulse-chase labeling of rRNA synthesis in wild-type strains 7 h after transfer to glucose medium. Cells were pulsed with [5,6-H]uracil for 1 min and then chased with a large excess of unlabeled uracil for the times indicated. (C) High molecular weight RNA analyzed on a 1.2% agarose/formaldehyde gel. (D) Low molecular weight RNA analyzed on a 6% polyacrylamide/urea gel. RNA species are labeled on the right of the panels. The asterisk in (C) indicates the position of the putative 5′ETS-D species.
Yeast pre-rRNA and processing
<b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal ... more <b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal subunit synthesis and for correct positioning of the mitotic spindle during mitosis"Nucleic Acids Research 2007;35(4):1354-1366.Published online 01 Feb 2007PMCID:PMC1849896.© 2007 The Author(s) () Structure of the yeast pre-rRNA, with locations of oligonucleotides used as hybridization probes. () Pre-rRNA processing pathway showing the intermediates detected by pulse-chase and northern analyses.
Cells form multiple spindles following Rrp14p depletion in spindle checkpoint mutants
<b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal ... more <b>Copyright information:</b>Taken from "Yeast Rrp14p is required for ribosomal subunit synthesis and for correct positioning of the mitotic spindle during mitosis"Nucleic Acids Research 2007;35(4):1354-1366.Published online 01 Feb 2007PMCID:PMC1849896.© 2007 The Author(s) Cells were treated as in . Tubulin was visualized by staining with rabbit anti-tubulin antibody and goat anti-rabbit coupled to FITC, in (panel b), (panel e), (panel h) and (panel k) cells 40 min after HU release. Nuclei were visualized by DAPI staining (panels a, d, g and j). Cell outlines are indicated with a dotted line (panels c, f, i and l).
Unraveling the function of m6A modification in acute myeloid leukemia
International Journal of Molecular Sciences, 2021
Growth and maturation of hematopoietic stem cells (HSCs) are largely controlled at both transcrip... more Growth and maturation of hematopoietic stem cells (HSCs) are largely controlled at both transcriptional and post-transcriptional levels. In particular, hematopoietic development requires a tight control of protein synthesis. Furthermore, translational deregulation strongly contributes to hematopoietic malignancies. Researchers have recently identified a new layer of gene expression regulation that consists of chemical modification of RNA species, which led to the birth of the epitranscriptomics field. RNA modifications provide an additional level of control in hematopoietic development by acting as post-transcriptional regulators of lineage-specific genetic programs. Other reviews have already described the important role of the N6-methylation of adenosine (m6A) within mRNA species in regulating hematopoietic differentiation and diseases. The aim of this review is to summarize the current status of the role of RNA modifications in the regulation of ribosome function, beyond m6A. In ...
Cell Death & Disease, 2021
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm caused by the presence of tyrosin... more 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 methyltransferase complex METTL3/METTL14 is upregulated in CML patients and that is required for proliferation of primary CML cells and CML cell lines sensitive and resistant to the TKI imatinib. We demonstrate that depletion of METTL3 strongly impairs global translation efficiency. In particular, our data show that METTL3 is crucial for the expression of genes involved in ribosome biogenesis and translation. Specifically, we found that METTL3 directly regulates the level of PES1 protein identified as an oncogene in several tumors. We propose a model in which nuclear METTL3/METTL14 methyltransferase complex modified nascent transcripts whos...
Cell Death & Disease, 2020
ABSTRACTEukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by ... more ABSTRACTEukaryotic Translation Initiation Factor 5A (EIF5A) is a translation factor regulated by hypusination, a unique posttranslational modification catalyzed by deoxyhypusine synthetase (DHPS) and deoxyhypusine hydroxylase (DOHH) starting from the polyamine spermidine. Emerging data are showing that hypusinated EIF5A regulates key cellular processes such as autophagy, senescence, polyamine homeostasis, energy metabolism, and plays a role in cancer. However, the effects of EIF5A inhibition in preclinical cancer models, the mechanism of action, and specific translational targets are still poorly understood. We show here that hypusinated EIF5A promotes growth of colorectal cancer (CRC) cells by directly regulating MYC biosynthesis at specific pausing motifs. Inhibition of EIF5A hypusination with the DHPS inhibitor GC7 or through lentiviral-mediated knockdown of DHPS or EIF5A reduces the growth of various CRC cells. Multiplex gene expression analysis reveals that inhibition of hypusi...
Cell Death & Disease, 2020
Long non-coding RNAs are emerging as new molecular players involved in many biological processes,... more Long non-coding RNAs are emerging as new molecular players involved in many biological processes, such as proliferation, apoptosis, cell cycle, migration, and differentiation. Their aberrant expression has been reported in variety of diseases. The aim of this study is the identification and functional characterization of clinically relevant lncRNAs responsible for the inhibition of miR-145-5p, a key tumor suppressor in thymic epithelial tumors (TETs). Starting from gene expression analysis by microarray in a cohort of fresh frozen thymic tumors and normal tissues, we identified LINC00174 as upregulated in TET. Interestingly, LINC00174 expression is positively correlated with a 5-genes signature in TETs. Survival analyses, performed on the TCGA dataset, showed that LINC00174 and its associated 5-genes signature are prognostic in TETs. Specifically, we show that LINC00174 favors the expression of SYBU, FEM1B, and SCD5 genes by sponging miR-145-5p, a well-known tumor suppressor microRN...
ABSTRACTMicrosatellite expansions of CCTG repeats in the CNBP gene leads to accumulation of toxic... more ABSTRACTMicrosatellite expansions of CCTG repeats in the CNBP gene leads to accumulation of toxic RNA and have been associated to DM2. However, it is still unclear whether the dystrophic phenotype is also linked to CNBP decrease, a conserved CCHC-type zinc finger RNA binding protein that regulates translation and is required for mammalian development.Here we show that depletion of Drosophila CNBP in muscles causes age-dependent locomotor defects that are correlated with impaired polyamine metabolism. We demonstrate that the levels of ornithine decarboxylase (ODC) and polyamines are significantly reduced upon dCNBP depletion. Of note, we show a reduction of the CNBP-polyamine axis in muscle from DM2 patients. Mechanistically, we provide evidence that dCNBP controls polyamine metabolism through binding dOdc mRNA and regulating its translation. Remarkably, the locomotor defect of dCNBP-deficient flies is rescued by either polyamine supplementation or dOdc1 overexpression. We suggest th...
Blood Advances, 2019
Key Points RA synergizes with the N-glycosylation inhibitor tunicamycin and ATO to induce AML cel... more Key Points RA synergizes with the N-glycosylation inhibitor tunicamycin and ATO to induce AML cell death via generation of ER and oxidative stress.
Nature Precedings, 2008
HP1 is a well known conserved protein involved in heterochromatin formation and gene silencing in... more HP1 is a well known conserved protein involved in heterochromatin formation and gene silencing in different species including humans1-4. A general model has been proposed for heterochromatin formation and epigenetic gene silencing in different species that implies an essential role for HP1. According to the model, histone methyltransferase enzymes (HMTases) methylate the histone H3 at lysine 9 (H3-MeK9), creating selective binding sites for itself and the chromodomain of HP15. This complex is thought to form a higher order chromatin state that represses gene activity. It has also been found that HP1 plays a role in telomere capping6. Surprisingly, recent data have suggested an association of HP1 in gene activity7-10 but the nature of this interaction is still completely obscure. Here we show, that HP1 is required for positive regulation of more than one hundred euchromatic genes by its association with the corresponding RNA transcripts and by its interaction with the well known prot...
Frontiers in Cell and Developmental Biology, 2019
RNA chemical modifications in coding and non-coding RNAs have been known for decades. They are ge... more RNA chemical modifications in coding and non-coding RNAs have been known for decades. They are generally installed by specific enzymes and, in some cases, can be read and erased by other specific proteins. The impact of RNA chemical modifications on gene expression regulation and the reversible nature of some of these modifications led to the birth of the word epitranscriptomics, in analogy with the changes that occur on DNA and histones. Among more than 100 different modifications identified so far, most of the epitranscriptomics studies focused on the N 6 -methyladenosine (m 6 A), which is the more abundant internal modification in protein coding RNAs. m 6 A can control several pathways of gene expression, including spicing, export, stability, and translation. In this review, we describe the interplay between m 6 A and non-coding RNAs, in particular microRNAs and lncRNAs, with examples of its role in gene expression regulation. Finally, we discuss its relevance in cell development and disease.
Cell Death & Disease, 2018
The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute... more The Wilms tumor 1 (WT1)-associated protein (WTAP) is upregulated in many tumors, including, acute myeloid leukemia (AML), where it plays an oncogenic role by interacting with different proteins involved in RNA processing and cell proliferation. In addition, WTAP is also a regulator of the nuclear complex required for the deposition of N 6methyladenosine (m6A) into mRNAs, containing the METTL3 methyltransferase. However, it is not clear if WTAP may have m6A-independent regulatory functions that might contribute to its oncogenic role. Here, we show that both knockdown and overexpression of METTL3 protein results in WTAP protein upregulation, indicating that METTL3 levels are critical for WTAP protein homeostasis. However, we show that WTAP upregulation is not sufficient to promote cell proliferation in the absence of a functional METTL3. Therein, these data indicate that the reported oncogenic function of WTAP is strictly connected to a functional m6A methylation complex.
Regenerative medicine research, 2017
MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to ... more MiR-204 and 211 enforced expression in murine mesenchymal stromal cells (MSCs) has been shown to induce adipogenesis and impair osteogenesis, through RUNX2 down-modulation. This mechanism has been suggested to play a role in osteoporosis associated with obesity. However, two further fundamental MSC functions, chondrogenesis and hematopoietic supporting activity, have not yet been explored. To this end, we transduced, by a lenti-viral vector, miR-204 and 211 in a model primary human MSC line, opportunely chosen among our MSC collection for displaying all properties of canonical bone marrow MSCs, except adipogenesis. Enforced expression of miR-204&211 in these cells, rescued adipogenesis, and inhibited osteogenesis, as previously reported in murine MSCs, but, surprisingly, also damaged cartilage formation and hematopoietic supporting activity, which were never explored before. RUNX2 has been previously indicated as the target of miR-204&211, whose down modulation is responsible for th...
Molecular cell, Jan 6, 2017
Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still larg... more Circular RNAs (circRNAs) constitute a family of transcripts with unique structures and still largely unknown functions. Their biogenesis, which proceeds via a back-splicing reaction, is fairly well characterized, whereas their role in the modulation of physiologically relevant processes is still unclear. Here we performed expression profiling of circRNAs during in vitro differentiation of murine and human myoblasts, and we identified conserved species regulated in myogenesis and altered in Duchenne muscular dystrophy. A high-content functional genomic screen allowed the study of their functional role in muscle differentiation. One of them, circ-ZNF609, resulted in specifically controlling myoblast proliferation. Circ-ZNF609 contains an open reading frame spanning from the start codon, in common with the linear transcript, and terminating at an in-frame STOP codon, created upon circularization. Circ-ZNF609 is associated with heavy polysomes, and it is translated into a protein in a s...
Oncotarget, 2016
Alterations in genetic programs required for terminal myeloid differentiation and aberrant prolif... more Alterations in genetic programs required for terminal myeloid differentiation and aberrant proliferation characterize acute myeloid leukemia (AML) cells. Here, we identify the host transcript of miR-223, linc-223, as a novel functional long noncoding RNA (lncRNA) in AML. We show that from the primary nuclear transcript, the alternative production of miR-223 and linc-223 is finely regulated during monocytic
Mirna, Sirna and Use Thereof in Therapy
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Papers by Alessandro Fatica