Resources

Publication

A noncanonical function of EIF4E limits ALDH1B1 activity and increases susceptibility to ferroptosis

Xin Chen, Jun Huang, Chunhua Yu, Jiao Liu, Wanli Gao, Jingbo Li, Xinxin Song, Zhuan Zhou, Changfeng Li, Yangchun Xie, Guido Kroemer, Jinbao Liu, Daolin Tang & Rui Kang

Ferroptosis is a type of lipid peroxidation-dependent cell death that is emerging as a therapeutic target for cancer. However, the mechanisms of ferroptosis during the generation and detoxification of lipid peroxidation products remain rather poorly defined. Here, we report an unexpected role for the eukaryotic translation initiation factor EIF4E as a determinant of ferroptotic sensitivity by controlling lipid peroxidation. A drug screening identified 4EGI-1 and 4E1RCat (previously known as EIF4E-EIF4G1 interaction inhibitors) as powerful inhibitors of ferroptosis. Genetic and functional studies showed that EIF4E (but not EIF4G1) promotes ferroptosis in a translation-independent manner. Using mass spectrometry and subsequent protein-protein interaction analysis, we identified EIF4E as an endogenous repressor of ALDH1B1 in mitochondria. ALDH1B1 belongs to the family of aldehyde dehydrogenases and may metabolize the aldehyde substrate 4-hydroxynonenal (4HNE) at high concentrations. Supraphysiological levels of 4HNE triggered ferroptosis, while low concentrations of 4HNE increased the cell susceptibility to classical ferroptosis inducers by activating the NOX1 pathway. Accordingly, EIF4E-dependent ALDH1B1 inhibition enhanced the anticancer activity of ferroptosis inducers in vitro and in vivo. Our results support a key function of EIF4E in orchestrating lipid peroxidation to ignite ferroptosis.

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Bioinformatic analysis

RiboSeq BioIT guideline (for Ribosome Proliling All In One Set, RiboSeq XL All In One Set, LACEseq kit)

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Publication

Deciphering the immunopeptidome in vivo reveals new tumour antigens

Alex M. Jaeger, Lauren E. Stopfer, Ryuhjin Ahn, Emma A. Sanders, Demi A. Sandel, William A. Freed-Pastor, William M. Rideout III, Santiago Naranjo, Tim Fessenden, Kim B. Nguyen, Peter S. Winter, Ryan E. Kohn, Peter M. K. Westcott, Jason M. Schenkel, Sean-Luc Shanahan, Alex K. Shalek, Stefani Spranger,Forest M. White & Tyler Jacks

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I) molecules. Current approaches to profling of MHC-I-associated peptides, collectively known as the immunopeptidome, are limited to in vitro investigation or bulk tumour lysates, which limits our understanding of cancer-specifc patterns of antigen presentation in vivo6. To overcome these limitations, we engineered an inducible afnity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the KrasLSL-G12D/+Trp53f/f mouse model (KP/Kb Strep). This approach enabled us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma and from lung adenocarcinoma (LUAD) in vivo. In addition, we profled the LUAD immunopeptidome from the alveolar type 2 cell of origin up to late-stage disease. Diferential peptide presentation in LUAD was not predictable by mRNA expression or translation efciency and is probably driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo induced CD8+T cell responses in naive mice and tumour-bearing mice. Many peptides specifc to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, which prompts the reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance8. Beyond cancer, the Kb Strep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease and autoimmunity

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Webinar and Podcast

Immagina biotech - part of Technicon spa

26.05-2022 - It's been quite a morning for us here at Immagina - We are pleased to announce that we are now part of Technicon spa, a holding with multiple new tech companies - including biotech - all over the world.

Review

White Paper RiboLace: Discover stories about translation that mRNA alone can’t tell.

RiboLace technology may be the secret you have been looking for to reveal new insights into ribosome profiling and translational research.

We have a new white paper that will explain how RiboLace can help you:

• Isolate and purify active ribosomes and ribosome-protected fragments, while leaving behind inactive ribosomes
• Simplify your ribosome profiling workflow
• Profile ribosome activity even with challenging samples or low sample input
• Profile disomes together with standard monosomes, if coupled with a library preparation developed in-house (available upon request as a service)

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Publication

Effective therapy for AML with RUNX1 mutation by cotreatment with inhibitors of protein translation and BCL2

Christopher P Mill, Warren Fiskus, Courtney D DiNardo, Christine Birdwell, John A Davis, Tapan M Kadia, Koichi Takahashi, Nicholas Short, Naval Daver, Maro Ohanian, Gautam Borthakur, Steven M Kornblau, Michael R Green, Yuan Qi, Xiaoping Su , Joseph D Khoury , Kapil N Bhalla

The majority of RUNX1 mutations in acute myeloid leukemia (AML) are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy, AML patients expressing mtRUNX1 exhibit inferior clinical outcome than those without mutant RUNX1. Studies presented here demonstrate that as compared with AML cells lacking mtRUNX1, their isogenic counterparts harboring mtRUNX1 display impaired ribosomal biogenesis and differentiation, as well as exhibit reduced levels of wild-type RUNX1, PU.1, and c-Myc. Compared with AML cells with only wild-type RUNX1, AML cells expressing mtRUNX1 were also more sensitive to the protein translation inhibitor homoharringtonine (omacetaxine) and BCL2 inhibitor venetoclax. Homoharringtonine treatment repressed enhancers and their BRD4 occupancy and was associated with reduced levels of c-Myc, c-Myb, MCL1, and Bcl-xL. Consistent with this, cotreatment with omacetaxine and venetoclax or BET inhibitor induced synergistic in vitro lethality in AML expressing mtRUNX1. Compared with each agent alone, cotreatment with omacetaxine and venetoclax or BET inhibitor also displayed improved in vivo anti-AML efficacy, associated with improved survival of immune-depleted mice engrafted with AML cells harboring mtRUNX1. These findings highlight superior efficacy of omacetaxine-based combination therapies for AML harboring mtRUNX1.

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Publication

Profiling and functional characterization of maternal mRNA translation during mouse maternal-to-zygotic transition

Chunxia Zhang, Meng Wang, Yisi Li, Yi Zhang

Translational regulation plays an important role in gene expression and function. Although the transcriptional dynamics of mouse preimplantation embryos have been well characterized, the global mRNA translation landscape and the master regulators of zygotic genome activation (ZGA) remain unknown. Here, by developing and applying a low-input ribosome profiling (LiRibo-seq) technique, we profiled the mRNA translation landscape in mouse preimplantation embryos and revealed the translational dynamics during mouse preimplantation development. We identified a marked translational transition from MII oocytes to zygotes and demonstrated that active translation of maternal mRNAs is essential for maternal-to-zygotic transition (MZT). We further showed that two maternal factors, Smarcd2 and Cyclin T2, whose translation is activated in zygotes, are required for chromatin reprogramming and ZGA, respectively. Our study thus not only filled in a knowledge gap on translational regulation during mammalian preimplantation development but also revealed insights into the critical function of maternal mRNA translation in MZT.

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Publication

Phospho-RNA sequencing with circAID-p-seq

Alessia Del Piano, Tea Kecman, Michael Schmid, Ruggero Barbieri, Luciano Brocchieri, Silvia Tornaletti, Claudia Firrito, Luca Minati, Paola Bernabò, Ilaria Signoria, Fabio Lauria, Thomas H Gillingwater, Gabriella Viero, Massimiliano Clamer

Most RNA footprinting approaches that require ribonuclease cleavage generate RNA fragments bearing a phosphate or cyclic phosphate group at their 3′ end. Unfortunately, current library preparation protocols rely only on a 3′ hydroxyl group for adaptor ligation or poly-A tailing. Here, we developed circAID-p-seq, a PCR-free library preparation for selective 3′ phospho-RNA sequencing. As a proof of concept, we applied circAID-p-seq to ribosome profiling, which is based on sequencing of RNA fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast and highly efficient sequencing of phospho-RNA fragments from eukaryotic cells and tissues. We used circAID-p-seq to portray ribosome occupancy in transcripts, providing a versatile and PCR-free strategy to possibly unravel any endogenous 3′-phospho RNA molecules.

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Publication

Mistranslation Drives Alterations in Protein Levels and the Effects of a Synonymous Variant at the Fibroblast Growth Factor 21 Locus

Ali Bayoumi, Asmaa Elsayed, Shuanglin Han, Salvatore Petta, Leon A. Adams, Rocio Aller, Anis Khan, Carmelo García-Monzón, María Teresa Arias-Loste, Luca Miele, Olivier Latchoumanin, Shafi Alenizi, Rocio Gallego-Durán, Janett Fischer, Thomas Berg, Antonio Craxì, Mayada Metwally, Liang Qiao, Christopher Liddle, Hannele Yki-Järvinen, Elisabetta Bugianesi, Manuel Romero-Gomez, Jacob George, Mohammed Eslam

Fibroblast growth factor 21 (FGF21) is a liver-derived hormone with pleiotropic beneficial effects on metabolism. Paradoxically, FGF21 levels are elevated in metabolic diseases. Interventions that restore metabolic homeostasis reduce FGF21. Whether abnormalities in FGF21 secretion or resistance in peripheral tissues is the initiating factor in altering FGF21 levels and function in humans is unknown. A genetic approach is used to help resolve this paradox. The authors demonstrate that the primary event in dysmetabolic phenotypes is the elevation of FGF21 secretion. The latter is regulated by translational reprogramming in a genotype- and context-dependent manner. To relate the findings to tissues outcomes, the minor (A) allele of rs838133 is shown to be associated with increased hepatic inflammation in patients with metabolic associated fatty liver disease. The results here highlight a dominant role for translation of the FGF21 protein to explain variations in blood levels that is at least partially inherited. These results provide a framework for translational reprogramming of FGF21 to treat metabolic diseases.

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Webinar and Podcast

Maria Luisa Malosio - Translation Analysis and Cognitive Decay

Maria Luisa Malosio, Research Scientist at the National Research Council (CNR - Consiglio Nazionale di Ricerca) in Milan, Italy, talks about translation and its role in her research with metabolic condition (like Type-2 Diabetes) and cognitive decline (e.g. Alzheimer's disease)

Webinar and Podcast

Emiliano Ricci - Virus Replication and Translation Analysis

Emiliano Ricci, Group Leader at the Department of Biology and Modeling of the Cell at the École Normale Supérieure de Lyon In France, talks about translation and its role in his research on virus replication,

Webinar and Podcast

Nathan James - Translation Analysis and the Circadian Rhythm

Nathan James, Postdoctoral Scientist at MRC Laboratory of Molecular Biology in Cambridge, England, talks about translation and its role in his research on the circadian clock

Webinar and Podcast

Alex Jaeger - Translation Analysis and Cancer Research

Alex Jaeger, Postdoctoral Fellow at the Koch Institute for Integrative Cancer Research at the Massachusetts Institute of Technology, talks about translation analysis and its role in his cancer research

Webinar and Podcast

Pan Yi - Future of Biotechnology Research

It was enlightning to talk to Pan Yi from Sequanta Technologies on the future of biotechnology research and where she sees this industry going

Webinar and Podcast

Paola Falletta - translation analysis and breast cancer

It was a pleasure talking to Paola Falletta, Senior Postdoc at the Cancer Imaging Unit at I.R.C.C.S. Ospedale San Raffaele, Milan, Italy, about her research on triple negative breast cancer and translation analysis

Bioinformatic analysis

ONT short RNA sequencing

CircAidMe is a tool designed to analyze data generated with CircAID-p-seq for Oxford Nanopore Technologies.

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Webinar and Podcast

Massimiliano Clamer - mRNA and Ribosome Research

It was great being able to talk with Massimiliano Clamer, Founder and President of IMMAGINA Biotechnology, on his experience as a founder in the biotechnology industry and the research IMMAGINA is doing with mRNA and ribosomes, 

Webinar and Podcast

Francesca Rapino - tRNAs, ribosomes and cancer

It was inspiring being able to speak with Francesca Rapino, PI (Chercheur Qualifié-FNRS) and assistant professor at the Stem Cells-Cancer Signaling Laboratory at the University of Liège, Belgium, on her research with tRNAs, ribosomes and cancer.

Webinar and Podcast

Adrian Gabriel Torres - Translation Analysis

It was great being able to speak to Ribo-Omics Grant Challenge Winner Adrian Gabirel Torres, Research Associate at the Gene Translation Lab in the Institute for Research in Biomedicine in Barcelona, on the research he is doing with translation analysis

Webinar and Podcast

Simone Sidoli - de novo protein synthesis

It was a pleasure to have spoken to Simone Sidoli, Assistant Professor and PI of Sidoli Lab at the Albert Einstein College of Medicine, Ullmann Research Center for Health Sciences in New York about his research in proteomics and specifically de novo protein synthesis

Review

Translation and Translation Heterogenity

Review on Translation and Translation Heterogenity with up-to-date scientific publications

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Review

Phospho-RNA: The Hidden Layer of Transcriptomics

Review on Phospho-RNA: The Hidden Layer of Transcriptomics with up-to-date scientific publications

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Publication

Puromycin reactivity does not accurately localize translation at the subcellular level

SU Enam, B Zinshteyn, DH Goldman, M Cassani eLife (2020) 9 e60303

Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

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Review

Proteo-Translatomics: When proteomics and translatomics merge

Review on Proteo-Translatomics: When proteomics and translatomics merge, with up-to-date scientific publications

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Publication

Biofunctional Surfaces for Smart Entrapment of Polysomes

L Lunelli, L Marocchi, L Pasquardini, L Vanzetti et al. Appl. Sci. 2021, 11, 776

Protein synthesis is a central process in all cells, crucial for cell development and maintenance. Translational dysregulation, in fact, is associated with cancer or neurodegenerative diseases. A panel of surfaces and surface functionalizations were screened for their ability to entrap polysomes with the ultimate aim to set up smart biofunctional surfaces for the purification of nonlabelled polysomes and their associated mRNAs. As a proof-of-concept, prepurified ribosomes and polysomes were incubated on multiple functional surfaces and characterized by atomic force microscopy to assess number and morphology of entrapped polysomes

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Publication

Current review about design's impact on analytical achievements of magnetic graphene oxide nanocomposites

P. Dramou, S.L. Dahn, F. Wang, Y. Sun, Z. Song TrAC Trends in Analytical Chemistry 2021, in Press

Magnetic nanocomposites involving graphene oxide which is a two-dimensional nanomaterial are an actual trend in many device systems used for analytical purposes.

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Publication

Spinal Muscular Atrophy: In the Challenge Lies a Solution

B Wirth Trends in Neurosciences (2021) in Press

The path from gene discovery to therapy in spinal muscular atrophy (SMA) has been a highly challenging endeavor, but also led to one of the most successful stories in neurogenetics. In SMA, a neuromuscular disorder with an often fatal outcome until recently, with those affected never able to sit, stand, or walk, children now achieve these motoric abilities and almost age-based development when treated presymptomatically.

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Publication

Phospho-RNA sequencing with CircAID-p-seq

Alessia Del Piano, Ruggero Barbieri, Michael Schmid, Luciano Brocchieri, Silvia Tornaletti, Claudia Firrito, Luca Minati, Paola Bernabo, Ilaria Signoria, Fabio Lauria, Thomas H. Gillingwater, Gabriella Viero, Massimiliano Clamer.

Accurate positional information concerning ribosomes and RNA binding proteins with respect to their transcripts is important to understand the global regulatory network underlying protein and RNA fate in living cells. Most footprinting approaches generate RNA fragments bearing a phosphate or cyclic phosphate groups at their 3′ end. Unfortunately, all current protocols for library preparation rely only on the presence of a 3′ hydroxyl group.

"Here, we developed circAID-p-seq, a PCR-free library preparation for 3′ phospho-RNA sequencing. We applied circAID-p-seq to ribosome profiling, which produces fragments protected by ribosomes after endonuclease digestion. CircAID-p-seq, combined with the dedicated computational pipeline circAidMe, facilitates accurate, fast, highly efficient and low-cost sequencing of phospho-RNA fragments from eukaryotic cells and tissues"

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Publication

Tunable Magnetic Hyperthermia Properties of Pristine and Mildly Reduced Graphene Oxide/Magnetite Nanocomposite Dispersions

E. Illés, E. Tombácz, Z. Hegedűs, T. Szabó et al. Nanomaterials 2020, 10, 2426

We present a study on the magnetic hyperthermia properties of graphene oxide/magnetite (GO/MNP) nanocomposites to investigate their heat production behavior upon the modification of the oxidation degree of the carbonaceous host.

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Publication

Improved thermal and mechanical properties of bismaleimide nanocomposites via incorporation of a new allylated siloxane graphene oxide

H Jiang, Z Li, J Gan, L Wang, Y Li RSC Adv. 2020, 10, 36853-36861.

A thermosetting resin system based on bismaleimide (BMI) has been developed via copolymerization with 4, 4′-diaminodiphenylsulfone in the presence of a newly synthesized graphene oxide, modified using allylated siloxane (AS-GO).

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Publication

SMN-primed ribosomes modulate the translation of transcripts related to Spinal Muscular Atrophy

Nat Cell Biol. 10, (2020) 1239-1251 Fabio Lauria, Paola Bernabò, Toma Tebaldi, Ewout Joan Nicolaas Groen, Elena Perenthaler, Federica Maniscalco, Annalisa Rossi, Deborah Donzel, Massimiliano Clamer, Marta Marchioretto, Neža Omersa, Julia Orri, Mauro Dalla Serra, Gregor Anderluh, Alessandro Quattrone, Alberto Inga, Thomas Henry Gillingwater, Gabriella Viero.

Survival motor neuron (SMN) protein—the loss of which causes the neuromuscular disease spinal muscular atrophy (SMA)—binds to ribosomes and that this interaction is tissue-dependent. SMN-primed ribosomes are preferentially positioned within the first five codons of a set of mRNAs that are enriched for translational enhancer sequences in the 5′ untranslated region (UTR) and rare codons at the beginning of their coding sequence.

“We investigated the association of SMN with active ribosomes in cellulo using the RiboLace method3, which facilitates the specific isolation of actively translating ribosomes.”

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Publication

Local mRNA translation in long-term maintenance of axon health and function

E Kim, H Jung Current Opinion in Neurobiology (2020) 63 15-22

Distal axons, remote from their cell bodies and nuclei, must survive the lifetime of an organism. Recent studies have provided compelling evidence that proteins are locally synthesized in healthy, mature central nervous system axons and presynaptic terminals in vivo.

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Publication

Active Ribosome Profiling with RiboLace

Cell Reports 25, (2018) 1097–1108 Massimiliano Clamer, Toma Tebaldi, Fabio Lauria, Paola Bernabo, Rodolfo F. Go mez-Biagi, Marta Marchioretto, Divya T. Kandala, Luca Minati, Elena Perenthaler, Daniele Gubert, Laura Pasquardini, Graziano Guella, Ewout J. N. Groen, Thomas H. Gillingwater, Alessandro Quattrone, Gabriella Viero.

Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation.

“We developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an anti-body-free and tag-free pull-down approach.”

Publication

The science of puromycin: From studies of ribosome function to applications in biotechnology

R Aviner Comput Struct Biotechnol J. 2020; 18 1074–1083.

Puromycin is a naturally occurring aminonucleoside antibiotic that inhibits protein synthesis by ribosome-catalyzed incorporation into the C-terminus of elongating nascent chains, blocking further extension and resulting in premature termination of translation.

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Publication

One-shot analysis of translated mammalian lncRNAs with AHARIBO

bioRxiv (2020) https://doi.org/10.1101/2020.04.20.050062 Luca Minati, Claudia Firrito, Alessia Del Piano, Alberto Peretti, Simone Sidoli, Daniele Peroni, Romina Belli, Francesco Gandolfi, Alessandro Romanel, Paola Bernabò, Jacopo Zasso, Alessandro Quattrone, Graziano Guella, Fabio Lauria, Gabriella Viero and Massimiliano Clamer

A vast portion of the mammalian genome is transcribed as long non-coding RNAs (lncRNAs) acting in the cytoplasm with largely unknown functions. Surprisingly, lncRNAs have been shown to interact with ribosomes, encode uncharacterized proteins, or act as ribosome sponges. These functions still remain mostly undetected and understudied owing to the lack of efficient tools for genome-wide simultaneous identification of ribosome-associated lncRNAs and peptide-producing lncRNAs.

“Here we present AHARIBO, a method for the detection of lncRNAs either untranslated, but associated with ribosomes, or encoding small peptides. Using AHARIBO in mouse embryonic stem cells during neuronal differentiation, we isolated ribosome-protected RNA fragments, translated RNAs and corresponding de novo synthesized polypeptides.”

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Publication

Recent advances in ribosome profiling for deciphering translational regulation

Y Wang, H Zhang, J Lu Methods (2020) 176 46-54

Messenger RNA (mRNA) translation is a central step of gene expression. Translational regulation plays essential roles in various biological processes. Ribosome profiling is a powerful technique to study genome-wide translational regulation at a single-codon resolution. Based on conventional ribosome profiling procedures, many new experimental methods have recently been developed to investigate the biology of mRNA translation from different aspects.

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Publication

Whole brain delivery of an instability-prone Mecp2 transgene improves behavioral and molecular pathological defects in mouse models of Rett syndrome

M. Luoni, S. Giannelli, M.T. Indrigo, A. Niro, L Massimino et al. eLife (2020) 9 e52629

Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization.

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Publication

Graphene Oxide Nanocomposite Magnetic Microbeads for the Remediation of Positively Charged Aromatic Compounds

Dalton Trans. 49 (2020) 3333-3340 Luca Minati, Giorgio Speranza, Viktor Micheli, Mauro Dalla Serra, Massimiliano Clamer

Integrating graphene as an inorganic nanostructure within a hydrogel matrix enables the creation of an unique hybrid composite combining the peculiar chemical and physical properties of graphene with the high porosity and stability of hydrogels.

“We report the synthesis of hybrid superparamagnetic hydrogel-based graphene oxide micro-beads for the adsorption and separation of positively charged aromatic molecule”

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Publication

SRSF7 maintains its homeostasis through the expression of Split-ORFs and nuclear body assembly

V Königs, COF Machado, B Arnold, N Blümel et al. Nature Structural & Molecular Biology (2020) 27 260–273

SRSF7 is an essential RNA-binding protein whose misexpression promotes cancer. Here, we describe how SRSF7 maintains its protein homeostasis in murine P19 cells using an intricate negative feedback mechanism. SRSF7 binding to its premessenger RNA promotes inclusion of a poison cassette exon and transcript degradation via nonsense-mediated decay (NMD). However, elevated SRSF7 levels inhibit NMD and promote translation of two protein halves, termed Split-ORFs, from the bicistronic SRSF7-PCE transcript.

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Publication

uORF-Tools—Workflow for the determination of translation-regulatory upstream open reading frames

A Scholz, F Eggenhofer, R Gelhausen, B Grüning et al. PLoS ONE (2019) 14 e0222459

Ribosome profiling (ribo-seq) provides a means to analyze active translation by determining ribosome occupancy in a transcriptome-wide manner. The vast majority of ribosome protected fragments (RPFs) resides within the protein-coding sequence of mRNAs. However, commonly reads are also found within the transcript leader sequence (TLS) (aka 5’ untranslated region) preceding the main open reading frame (ORF), indicating the translation of regulatory upstream ORFs (uORFs).

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Publication

Targeting Translation Activity at the Ribosome Interface with UV-Active Small Molecules

ACS Omega 4, (2019) 10336–10345
Divya T. Kandala, Alessia Del Piano, Luca Minati, and Massimiliano Clamer

Puromycin is a well-known antibiotic that is used to study the mechanism of protein synthesis and to monitor ribosome activity due to its incorporation into nascent peptide chains. However, puromycin effects outside the ribosome catalytic core remain unexplored.

“Here, we developed two analogues (3PB and 3PC) of the 3′-end of tyrosylated-tRNA that can efficiently interact with several proteins associated with ribosomes. We biochemically characterized the binding of these analogues and globally mapped the direct small molecule–protein interactions in living cells using clickable and photoreactive puromycin-like probes in combination with in-depth mass spectrometry.”

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Bioinformatic analysis

GitHub pipeline for RiboSeq analysis (RiboLace Mod. 2 kit ONLY!)

BioIT resource: ILMN ribosome profiling - data analysis with codes

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