Molteplici funzioni della proteina “Fragile X mental retardation Protein” nella regolazione dell’espressione genica

La proteina FMRP (Fragile X Mental Retardation Protein) è la causa principale della Sindrome dell’X Fragile, la più comune forma di ritardo mentale ereditario. FMRP è una proteina che lega gli RNA e può legare direttamente gli mRNA attraverso elementi o sequenze in cis (AU-rich sequences, G-quartet elements, kissing-complex) o indirettamente attraverso piccoli RNA non codificanti come BC1 o i microRNA. FMRP regola il metabolismo degli mRNA ed ha un ruolo importante nella traduzione di un messaggero e nella localizzazione di un mRNA ai dendriti. Durante il mio dottorato, ho studiato i possibili e molteplici meccanismi di FMRP nel regolare l’espressione genica. Nella prima parte della tesi, ho descritto un terza e nuova funzione regolatoria di FMRP nel controllare la stabilità di un mRNA. In topo, ho osservato che FMRP lega, in vivo, l’mRNA codificante per PSD-95, una molecola chiave che regola l’apprendimento e l’efficienza del segnale sinaptico. L’interazione si verifica nella regione 3’ non tradotta dell’mRNA di PSD-95 e permette d’incrementare la stabilità del messaggero. Inoltre, la stabilità è regolata in seguito all’attivazione dei recettori metabotropici del glutammato. Il controllo nella stabilità del mRNA di PSD-95 si verifica solo nell’ippocampo, questo risultato mostra per la prima volta che FMRP ha uno ruolo regolatorio in una specifica regione del cervello. Sebbene, ho osservato che l’mRNA di PSD-95 è sinapticamente localizzato in vivo, la localizzazione si verifica indipendentemente da FMRP. Attraverso questo studio, ho osservato che la regolazione della stabilità di un mRNA può contribuire a deficit cognitivi nei soggetti affetti dalla Sindrome dell’X Fragile. Nella seconda parte, ho descritto il possibile ruolo di FMRP nel pathway dei microRNA. I miRNA regolano l’espressione genica ed FMRP è stata trovata associata al complesso RISC. Tuttavia, la presenza e la funzione di questo complesso non è stata ancora descritta nel cervello. Nel mio laboratorio, prime evidenze sperimentali hanno osservato che FMRP non regola la maturazione e la distribuzione polisomale dei miRNA nel cervello. In seguito, ho eseguito uno studio traduzionale in cellule neuronali WT e Fmr1 KO trasfettate con un gene reporter per la EGFP che possiede dei siti di legame per let7c. L’analisi ha mostrato che FMRP non è coinvolto nella inibizione dell’espressione genica mediata da FMRP. Un’analisi biochimica mi hanno permesso di osservare che FMRP immunoprecipita con Ago2 in cervelli murini, ma i miRNA non sono presenti nello stesso complesso. Questo studio ha mostrato la presenza di un nuovo complesso dove FMRP e Ago2 potrebbero regolare specifici messaggeri con un pathway indipendente dai microRNA.The Fragile X Mental Retardation Protein (FMRP) is the main cause of the Fragile X Syndrome, the most common form of inherited mental retardation. FMRP is an RNA binding protein and it may bind mRNAs directly via cis elements or sequences (AU-rich sequences, G-quartet elements, kissing-complex) or indirectly using small non coding RNAs such as BC1 or microRNAs. FMRP regulates mRNA metabolism and it has important roles in mRNA translation and may be fundamental in mRNA localization to dendrites. During my PhD, I have studied the possible multiple mechanisms and functions of FMRP in the regulation of gene expression. In the first part of my thesis I have reported a third cytoplasmic regulatory function for FMRP: control of mRNA stability. In mice, I have found that FMRP binds, in vivo, the mRNA encoding PSD-95, a key molecule that regulates neuronal synaptic signaling and learning. This interaction occurs through the 3’untranslated region of the PSD-95 (also known as Dlg4) mRNA, increasing message stability. Moreover, stabilization is further increased by mGluR activation. The control of PSD-95 mRNA stability occours only in the hippocampus, this result shows for the first time that FMRP has a specific brain-region role. Although, I have also found that the PSD-95 mRNA is synaptically localized in vivo, localization occurs independently of FMRP. Through my functional analysis of this FMRP target I provide evidence that dysregulation of mRNA stability may contribute to the cognitive impairments in individuals with FXS. The second part describes the possible role of FMRP in the miRNAs pathway. miRNAs regulate the gene expression and FMRP is associated with component of the RISC complex. Yet, the presence and the function of the putative complex is unknown in brain. In my lab, previous evidences have shown that FMRP does not regulate miRNAs maturation/stability and the polysome/mRNP distribution of miRNAs in brain. I have performed a translational assay in both WT and FMR1 KO neuronal cells following transfection of a EGFP construct harbouring a synthetic Responsive Element for let7c. The analysis has shown that FMRP is not involved in let7c mediated inhibition of gene expression. I have found that FMRP immunoprecipitates with Ago2 in mice brain but miRNAs are not present in the same complex. The study has shown the presence of a complex where FMRP and Ago2 could regulate specific mRNAs with an indipendent miRNAs pathway

NoRC Recruitment by H2A.X Deposition at rRNA Gene Promoter Limits Embryonic Stem Cell Proliferation

Summary: Embryonic stem cells (ESCs) display an abbreviated cell cycle, resulting in a short doubling time and rapid proliferation. The histone variant H2A.X is critical for proliferation of stem cells, although mechanistic insights have remained obscure. Here, we show that H2A.X defines the rate of mouse ESC proliferation independently of the DNA damage response pathway, and it associates with three major chromatin-modifying complexes. Our functional and biochemical analyses demonstrate that H2A.X-associated factors mediate the H2A.X-dependent effect on ESC proliferation and involve the nucleolar remodeling complex (NoRC). A specific H2A.X deposition at rDNA promoters determines the chromatin recruitment of the NoRC, histone modifications, the rRNA transcription, and the rate of proliferation. Collectively, our results suggest that NoRC assembly by H2A.X deposition at rRNA promoters silences transcription, and this represents an important regulatory component for ESC proliferation. : Histone variant H2A.X defines the rate of embryonic stem cell proliferation. Eleuteri et al. identify H2A.X-interacting proteins, and they show that H2A.X deposition at rDNA promoters assembles the NoRC, which represses rRNA transcription and determines the rate of self-renewal. Keywords: ribosomal biogenesis, rRNA, rDNA, stem cells, TIP5, SNF2H, SPT16, BRG1, H2A.X, G1, cell cycle, cell cycle arrest, proliferatio

A new function for the fragile X mental retardation protein in regulation of PSD-95 mRNA stability

Fragile X syndrome ( FXS) results from the loss of the fragile X mental retardation protein ( FMRP), an RNA- binding protein that regulates a variety of cytoplasmic mRNAs. FMRP regulates mRNA translation and may be important in mRNA localization to dendrites. We report a third cytoplasmic regulatory function for FMRP: control of mRNA stability. In mice, we found that FMRP binds, in vivo, the mRNA encoding PSD- 95, a key molecule that regulates neuronal synaptic signaling and learning. This interaction occurs through the 3' untranslated region of the PSD- 95 ( also known as Dlg4) mRNA, increasing message stability. Moreover, stabilization is further increased by mGluR activation. Although we also found that the PSD- 95 mRNA is synaptically localized in vivo, localization occurs independently of FMRP. Through our functional analysis of this FMRP target we provide evidence that dysregulation of mRNA stability may contribute to the cognitive impairments in individuals with FXS.status: publishe

The fragile X syndrome protein represses activity-dependent translation through CYFIP1, a new 4E-BP

Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.status: publishe

A new function for the fragile X mental retardation protein in regulation of PSD-95 mRNA stability

Fragile X syndrome ( FXS) results from the loss of the fragile X mental retardation protein ( FMRP), an RNA- binding protein that regulates a variety of cytoplasmic mRNAs. FMRP regulates mRNA translation and may be important in mRNA localization to dendrites. We report a third cytoplasmic regulatory function for FMRP: control of mRNA stability. In mice, we found that FMRP binds, in vivo, the mRNA encoding PSD- 95, a key molecule that regulates neuronal synaptic signaling and learning. This interaction occurs through the 3' untranslated region of the PSD- 95 ( also known as Dlg4) mRNA, increasing message stability. Moreover, stabilization is further increased by mGluR activation. Although we also found that the PSD- 95 mRNA is synaptically localized in vivo, localization occurs independently of FMRP. Through our functional analysis of this FMRP target we provide evidence that dysregulation of mRNA stability may contribute to the cognitive impairments in individuals with FXS

Proceedings Of The 23Rd Paediatric Rheumatology European Society Congress: Part Two

PubMe