The localization of messenger RNAs (mRNAs) allows a cell to restrict certain proteins to specific subcellular domains. In neurons, this process critically contributes to long-term synaptic plasticity and thereby to higher brain functions such as learning and memory. Key components of the mRNA transport machinery, so-called trans-acting factors, are conserved across species including the RNA-binding protein Staufen (Stau), the zipcode binding proteins (ZBPs) and the heterogeneous nuclear ribonucleoproteins (hnRNPs). One key feature of mRNA localization is that the transcript has to be kept translationally silent during its transport towards its target compartment. As a consequence, RNA transport and control of translation are thought to be tightly coupled. In many cases, the loss of one component of the transport machinery or the misexpression of a specific mRNA causes abnormalities during neuronal development, for example in axonal and dendritic outgrowth or in the development of dendritic spines. These phenotypes have recently been correlated with different neurological disorders such as Down’s syndrome, Rett syndrome and fragile X mental retardation syndrome as well as spinal muscular atrophy.
Our group is interested in understanding the molecular mechanisms of RNA localization in neurons. We want to identify which proteins bind to and transport the mRNAs into dendrites and deliver them to synapses. To study this question, we combine techniques in molecular, cell and neurobiology with fluorescence and time-lapse video-microscopy to visualize protein and RNA movements in living cells. This multidisciplinary approach will help us to unravel the mechanisms that regulate both RNA and RNA-binding proteins during localization.
This knowledge will be crucial for a better understanding of the pathogenesis of neurological diseases. Specific projects are:
- Characterization of Pumilio (Pum) proteins in mammalian neurons. We have recently identified Pum2 as a new component of the RNA transport and translational machinery in neurons. We are currently investigating the function of Pum proteins in neurons and their role in mRNA transport and translational control.
- Molecular composition of translationally repressed ribonucleoprotein particles (RNPs). To understand how mRNA transport and translational repression are coupled, we identify proteins and RNA components of dendritic RNPs using Pum and other trans-acting factors such as Stau. We also investigate whether components of these RNPs play a role in mRNA stability and/or degradation.
- Post-translational control of RNA-binding proteins. Phosphorylation and SUMOylation are post-translational modifications that have been shown to play important roles in regulating protein activity and turnover in dendrites and at synapses. In contrast to phosphorylation and SUMOylation, protein methylation is poorly understood in the context of RNA transport and synaptic activity.
Preliminary data suggest, however, that some RNA-binding proteins contain methylation sites. We will study whether methylation regulates mRNA transport and subsequent translation and how this effect might be exerted. This opens up a new and exiting field of investigation which we are actively engaged in.
- "Characterization of specific mRNA variants in pediatric myelodysplastic syndrome (MDS)"
Notable differences set myelodysplastic syndrome (MDS) in children apart from the group of malignant myeloid disorders bearing the same denominator in adults. First, the traditional classification of MDS in adults using cytomorphology and number of myeloblasts is of limited utility in children as far as treatment decisions and prognosis are concerned. Second, the clinical presentation of childhood MDS is heterogeneous and there is broad overlap with inherited bone marrow failure disorders making the differential diagnosis a challenging puzzle. Last but not least, the mutational landscape is composed of lesions other than those found in the elderly, supporting fundamental differences in pathogenesis. The rapid growth of RNA‑Sequencing (RNA-seq) provides the chance to observe variation in the genome at the exon level and screening of alternative splicing (AS) is one of its important applications. AS is an important mechanism that could result in proteins with different functions from one mRNA precursor and it has been shown to affect the status of numerous diseases.
The scientific motivation for our project is three fold. First, systematic analysis of the biological function of the multiple AS isoforms associated with particular pathways and biological processes in childhood MDS is still missing. Second, the role of AS in pediatric MDS is largely unexplored. Third, the combinatorial logic of AS regulation and its integration with other regulatory cellular circuits remains poorly understood. The main goal is to systematically explore the role of AS and its regulation in the initiation and progression of this disease.
The main expected consequence of this project is to increase our knowledge of gene regulation networks during initiation and progression of childhood MDS and provide an integral picture of mechanisms of AS control. In addition to generating data, our goal is to acquire true understanding of the molecular logic behind these processes. The project has the potential to shed light on current conundrums in the field of hematopoietic malignancies because: a) aims to carry out frontier research in the emerging area of AS in childhood MDS, b) represent ground-breaking efforts driven by ambitious, interdisciplinary goals that combine experimental and computational analysis using mechanistic, high-throughput and systems biology approaches that can provide a comprehensive and integrated view of AS regulation and c) has the potential to open new horizons and opportunities for research in the gene regulation, to identify factors and regulatory mechanisms of relevance for regenerative medicine and to identify novel players to understand tumor initiation and progression.
This project is generously supported by AIL Trento https://www.ailtrentino.it/
“Caratterizzazione delle varianti di mRNA specifici nelle sindromi mielodisplastiche pediatriche”
L'obiettivo principale di questo progetto di ricerca, reso possibile grazie al contributo di A.I.L. di Trento, è lo studio delle sindromi mielodisplastiche (SMD) in età pediatrica causate principalmente da danni nel DNA delle cellule staminali presenti all’interno del midollo osseo. Le cellule staminali danneggiate non riescono a produrre una quantità adeguata di cellule del sangue funzionali, causando una carenza di globuli rossi, globuli bianchi e piastrine. In circa un terzo di pazienti con SMD la malattia degenera in leucemia mieloide acuta (LMA). Le SMD sono patologie rare in età pediatrica (rappresentano solo il 4% di tutti i tumori del sangue pediatrici). Essendo rare come malattie, le SMD pediatriche, rimangono ancora oggi patologie di sottovalutata incidenza clinica e di insoddisfacente risoluzione terapeutica.
Il progetto è rivolto allo studio delle alterazioni del meccanismo di maturazione (splicing) dell’RNA nelle SMD pediatriche. Lo splicing è una fase della trascrizione in cui le diverse porzioni di RNA appena prodotto sono unite a formare l’RNA messaggero maturo (mRNA) da cui derivano le proteine. Grazie allo splicing, la cellula non solo può esercitare un accurato controllo sull’espressione genica, ma può anche aumentare la variabilità dell’informazione
- Paolo Macchi, PI
- Annalisa Rossi, postdoctoral fellow
- Lorena Zubovic, postdoctoral fellow
- Lisa Gasperini, postdoctoral fellow
- Ralf Dahm, Institute of Molecular Biology gGmbH (IMB), Mainz, Germany
- Stefano Ferrari, Institute of Molecular Cancer Research, University of Zurich, Switzerland
- Michael A. Kiebler, Institute for Cell Biology, Ludwig-Maximilians-Universität, München, Germany
- Philippe Monnier, University of Toronto/University Health Network, Toronto, Canada
- Stefan Tenzer, Institute of Immunology, Johannes Gutenberg-Universität, Mainz, Germany
- Jacqueline Trotter, Department of Molecular Cell Biology, University of Mainz, Germany
- John P. Vessey, Development and Stem Cell Biology, The Hospital for Sick Children - MaRS Centre Toronto Medical Discovery East Tower 101 College Street, Toronto, Canada
Stefan Tenzer, Albertomaria Moro, Jörg Kuharev, Ashwanth Christopher Francis, Laura Vidalino, Alessandro Provenzani and Paolo Macchi. Proteome-wide characterization of the RNA-binding protein RALY-interactome using the iBioPQ (in vivo-Biotinylation-Pulldown-Quant) approach. Journal of Proteome Research (2013) PMID 23614458
Michela A Denti, Gabriella Viero, Alessandro Provenzani, Alessandro Quattrone, and Paolo Macchi. mRNA fate: Life and death of the mRNA in the cytoplasm. RNA Biology 10(3) (2013) PMID 23466755
John P. Vessey, Lucia Schoderboeck, Ewald Gingl, Ettore Luzi, Julia Riefler, Francesca Di Leva, Daniela Karra, Michael A. Kiebler and Paolo Macchi. Mammalian Pum2 regulates dendrite morphogenesis and synaptic function. Proc. Natl. Acad. Sci. USA, 107: 3222-7.
Fabian Tübing, Georgia Vendra1, Martin Mikl, Paolo Macchi, Sabine Thomas and Michael A. Kiebler. Dendritically localized transcripts are sorted into distinct sRNPs that display fast directional motility along dendrites of hippocampal neurons. J. of Neuroscience, 30: 4160-70.
Zeitelhofer M, Karra D, Vessey JP, Jaskic E, Macchi P, Thomas S, Riefler J, Kiebler M, Dahm R. (2009). High-efficiency transfection of short hairpin RNAs-encoding plasmids into primary hippocampal neurons. J. Neurosci. Res. 87: 289-300.
Zeitelhofer M, Macchi P and Dahm R. (2008). Perplexing bodies: The putative roles of P-bodies in neurons. RNA Biol. 5: 244-8.
Vessey JP, Macchi P, Stein JM, Mikl M, Hawker KN, Vogelsang P, Wieczorek K, Vendra G, Riefler J, Tübing F, Aparicio SA, Abel T, Kiebler MA (2008). A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis. Proc Natl Acad Sci U S A, 105:16374-9.
Vessey J, Dahm R, Macchi P. Silence please: Pumilio speaks: translational silencing by Pumilio proteins. In “RNA Binding Proteins in Development and Disease”. Denman (Ed.), p. 75-105.
Dahm R, Kiebler M and Macchi P (2008). “mRNA localisation and local protein translation“. Methods in Cell Biology 85: 293-327.
Vessey JP, Vaccani A, Xie Y, Dahm R, Karra D, Kiebler MA, Macchi P. 2006. Dendritic localization of the translational repressor Pumilio 2 and its contribution to dendritic stress granules. J Neurosci 26: 6496-6508.
Massi P, Vaccani A, Bianchessi S, Costa B, Macchi P, Parolaro D. 2006. The non-psychoactive cannabidiol triggers caspase activation through the involvement of oxidative stress in human glioma cells. Cell Mol Life Sci 63: 2057-2066.
Martel C, Macchi P, Furic L, Kiebler MA, DesGroseillers L. 2005. Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants. Biochem J 393: 245-254.
Kiebler M, Peter-Jansen R, Dahm D, Macchi P. 2005. A putative nuclear function for mammalian Staufen. Trends Biochem Sci 30: 228-231.
Goetze B, Tuebing F, Xie Y, Thomas S, Macchi P, Kiebler MA. 2005. The brain specific double-stranded RNA binding protein Staufen2 is required for dendritic spine development. J Cell Biol 172: 221-231.
Macchi P, Brownawell AM, Grunewald B, Macara I, Kiebler M. 2004. The brain specific double-stranded RNA-binding protein Staufen2:
nucleolar accumulation and isoform specific Exportin-5 dependent export“. J Biol Chem 279.
Macchi P, Hemraj I, Grunewald B, Mallardo M, Goetze B, Kiebler M. 2003. A GFP-based system to uncouple mRNA transport from translation in a single living neuron. Mol Biol Cell 14: 1570-1582.
Macchi P, Kroening S, Palacios IM, Baldassa S, Grunewald B, Ambrosino C, Goetze B, Lupas A, St Johnston D, Kiebler M. 2003. Barentsz, a new component of the Staufen-containing ribonucleoprotein particles in mammalian cells, interacts with Staufen in a RNA-dependent manner. J Neurosci 23: 5778-5788.
Simons M, Kramer EM, Macchi P, Rathke-Hartlieb S, Trotter J, Kiebler M, Nave KA, Schulz J. 2002. Accumulation of the myelin proteolipid protein in the endosomal system impairs raft membrane trafficking: implication for Pelizaeus-Merzbacher disease. J Cell Biol 157: 327-336.
Monnier P, Sierra A, Macchi P, Deitinghoff L, Andersen JS, Mann M, Flad M, Hornberger M, Stahl B, Bonhoeffer F, Mueller B. 2002. RGM, a novel repulsive guidance molecule for retinal axons. Nature 419: 392-395.
Kiebler M, Macchi P. 2001. mRNA localization revisited. Cell 107: 269-272.
Macchi P, Villa A, Giliani S, Sacco MG, Frattini A, Porta F, Ugazio A, Johnston J, Candotti F, O' Shea J, Vezzoni P, Notarangelo G. 1995. Mutations of JAK3 gene in patients with autosomal severe combined immunodeficiency (SCID). Nature 377: 65-68.
Manuel Zeitelhofer, Daniela Karra, Paolo Macchi, Marco Tolino, Sabine Thomas, Martina Schwarz, Michael Kiebler and Ralf Dahm (2008). Dynamic interaction between P-bodies and transport RNPs in dendrites of mature hippocampal neurons. J. of Neuroscience, In press.
Zeitelhofer Manuel, Karra Daniela Vessey John, Jaskic Elmir, Macchi Paolo, Thomas Sabine, Riefler Julia, Kiebler Michael and Dahm Ralf (2008). Improved protocol for high-efficiency transfection of shRNA-encoding plasmids into primary hippocampal neurons. J. of Neuroscience Research, In press.
Xie Y, Vessey JP, Konecna A, Dahm R, Macchi P and Kiebler MA (2007). “The dendritic spine-associated GTPase Septin 7 is a regulator of dendrite branching and dendritic spine morphology”. Current Biology 17: 1746-51.
Dahm R, Kiebler K, Macchi P (2007). “RNA localization in the nervous system“. Seminars in Cell and Developmental Biology 18: 216-23.
Dahm R and Macchi P (2007). “Human pathologies associated with defective mRNA transport”. Biology of the Cell, 99: 649-61.