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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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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

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

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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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

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

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

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

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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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

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

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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