Catia Andreassi

An NGF-responsive element targets myo-inositol monophosphatase-1 mRNA to sympathetic neuron axons

mRNA localization is an evolutionary conserved mechanism that underlies the establishment of cellular polarity and specialized cell functions. To identify mRNAs localized in subcellular compartments of developing neurons, we took an original approach that combines compartmentalized cultures of rat sympathetic neurons and sequential analysis of gene expression (SAGE). Unexpectedly, the most abundant transcript in axons was mRNA for myo-inositol monophosphatase-1 (Impa1), a key enzyme that regulates the inositol cycle and the main target of lithium in neurons. A novel localization element within the 3′ untranslated region of Impa1 mRNA specifically targeted Impa1 transcript to sympathetic neuron axons and regulated local IMPA1 translation in response to nerve growth factor (NGF). Selective silencing of IMPA1 synthesis in axons decreased nuclear CREB activation and induced axonal degeneration. These results provide insights into mRNA transport in axons and reveal a new NGF-responsive localization element that directs the targeting and local translation of an axonal transcript.

Inositol pyrophosphates regulate JMJD2C-dependent histone demethylation

Significance Epigenetic modifications of chromatin are emerging as important regulatory mechanisms of many nuclear processes. Numerous proteins have been identified that mediate these modifications in a dynamic manner. However, less is known about the signaling pathways that transduce upstream signals into chromatin changes. Here, we show that the signaling molecule inositol pyrophosphate (IP7) synthesised by inositol hexakisphosphate kinase 1 plays a key role in regulating the association of one of these proteins, Jumonji domain containing 2C with chromatin, thereby controlling the levels of a number of crucial epigenetic modifications important to regulate gene expression. Epigenetic modifications of chromatin represent a fundamental mechanism by which eukaryotic cells adapt their transcriptional response to developmental and environmental cues. Although an increasing number of molecules have been linked to epigenetic changes, the intracellular pathways that lead to their activation/repression have just begun to be characterized. Here, we demonstrate that inositol hexakisphosphate kinase 1 (IP6K1), the enzyme responsible for the synthesis of the high-energy inositol pyrophosphates (IP7), is associated with chromatin and interacts with Jumonji domain containing 2C (JMJD2C), a recently identified histone lysine demethylase. Reducing IP6K1 levels by RNAi or using mouse embryonic fibroblasts derived from ip6k1−/− knockout mice results in a decreased IP7 concentration that epigenetically translates to reduced levels of trimethyl-histone H3 lysine 9 (H3K9me3) and increased levels of acetyl-H3K9. Conversely, expression of IP6K1 induces JMJD2C dissociation from chromatin and increases H3K9me3 levels, which depend on IP6K1 catalytic activity. Importantly, these effects lead to changes in JMJD2C-target gene transcription. Our findings demonstrate that inositol pyrophosphate signaling influences nuclear functions by regulating histone modifications.

3′ UTR Remodelling of Axonal Transcripts in Sympathetic Neurons

Asymmetric localization of mRNAs is a mechanism that constrains protein synthesis to subcellular compartments. In neurons, mRNA transcripts are transported to both dendrites and axons where they are rapidly translated in response to extracellular stimuli. To characterize the 3’ UTR isoforms localized in axons and cell bodies of sympathetic neurons we performed 3’ end-RNA sequencing. We discovered that isoforms transported to axons had significantly longer 3’ UTRs compared to cell bodies. Moreover, more than 100 short 3’ UTR isoforms were uniquely expressed in axons. Analysis of the long 3’ UTR of IMPA1 indicated that a multiprotein complex including Upf1, HuD and Ago2 mediated 3’ UTR cleavage. This event enhanced IMPA1 translation and was necessary for maintaining axon integrity. 3’ UTR cleavage took place in axons and was not limited to IMPA1 but extended to other transcripts with similar expression patterns. We conclude that the 3’ UTR of neuronal transcripts undergo post-transcriptional remodelling and describe an alternative mechanism that regulates local protein synthesis. HIGHLIGHTS 3’end-Seq reveals distinct expression of alternative 3’UTR isoforms in axons and cell bodies of sympathetic neurons. Short 3’UTR isoforms uniquely detected in axons are generated in situ by cleavage of longer precursors. A protein complex containing Upf1, HuD, Pabpc4 and Ago2 mediates the cleavage of 3’UTRs.Abstract The 3’ untranslated regions (3’UTRs) of messenger RNAs (mRNA) are non-coding sequences that regulate several aspects of mRNA metabolism, including intracellular localisation and translation. Here, we show that in sympathetic neuron axons, the 3’UTRs of many transcripts undergo cleavage, generating both translatable isoforms expressing a shorter 3’UTR, and 3’UTR fragments. 3’end RNA sequencing indicated that 3’UTR cleavage is a potentially widespread event in axons, which is mediated by a protein complex containing the endonuclease Ago2 and the RNA binding protein HuD. Analysis of the Inositol monophosphatase 1 (Impa1) mRNA revealed that a stem loop structure within the 3’UTR is necessary for Ago2 cleavage. Thus, remodeling of the 3’UTR provides an alternative mechanism that simultaneously regulates local protein synthesis and generates a new class of 3’UTR RNAs with yet unknown functions.

Proteomic analysis of S-nitrosylated nuclear proteins in rat cortical neurons

The S-nitrosylated proteome directs dendritogenesis in developing cortical neurons. Discovering the neuronal SNO-ome Posttranslational modifications (PTMs) regulate protein abundance, localization, and function. S-nitrosylation (or “SNO”) is a PTM involving the attachment of a nitric oxide group to target proteins. Smith et al. performed specialized proteomics on nuclear extracts from embryonic rat cortical neurons to identify hundreds of proteins that could be S-nitrosylated. They identified site-specific SNO sites for more than half of these proteins and showed that disrupting the S-nitrosylation of several of these impaired the outgrowth of neuronal dendrites in culture, a process that is critical for brain development and neuronal function in vivo. The findings provide a resource from which to explore the role of S-nitrosylation in neuronal development. Neurons modulate gene expression in response to extrinsic signals to enable brain development, cognition, and learning and to process stimuli that regulate systemic physiological functions. This signal-to-gene communication is facilitated by posttranslational modifications such as S-nitrosylation, the covalent attachment of a nitric oxide (NO) moiety to cysteine thiols. In the cerebral cortex, S-nitrosylation of histone deacetylase 2 (HDAC2) is required for gene transcription during neuronal development, but few other nuclear targets of S-nitrosylation have been identified to date. We used S-nitrosothiol resin-assisted capture on NO donor-treated nuclear extracts from rat cortical neurons and identified 614 S-nitrosylated nuclear proteins. Of these, 131 proteins have not previously been shown to be S-nitrosylated in any system, and 555 are previously unidentified targets of S-nitrosylation in neurons. The sites of S-nitrosylation were identified for 59% of the targets, and motifs containing single lysines were found at 33% of these sites. In addition, lysine motifs were necessary for promoting the S-nitrosylation of HDAC2 and methyl-CpG binding protein 3 (MBD3). Moreover, S-nitrosylation of the histone-binding protein RBBP7 was necessary for dendritogenesis of cortical neurons in culture. Together, our findings characterize S-nitrosylated nuclear proteins in neurons and identify S-nitrosylation motifs that may be shared with other targets of NO signaling.

Post-transcriptional Processing of mRNA in Neurons: The Vestiges of the RNA World Drive Transcriptome Diversity

Neurons are morphologically complex cells that rely on the compartmentalization of protein expression to develop and maintain their extraordinary cytoarchitecture. This formidable task is achieved, at least in part, by targeting mRNA to subcellular compartments where they are rapidly translated. mRNA transcripts are the conveyor of genetic information from DNA to the translational machinery, however, they are also endowed with additional functions linked to both the coding sequence (open reading frame, or ORF) and the flanking 5′ and 3′ untranslated regions (UTRs), that may harbor coding-independent functions. In this review, we will highlight recent evidences supporting new coding-dependent and -independent functions of mRNA and discuss how nuclear and cytoplasmic post-transcriptional modifications of mRNA contribute to localization and translation in mammalian cells with specific emphasis on neurons. We also describe recently developed techniques that can be employed to study RNA dynamics at subcellular level in eukaryotic cells in developing and regenerating neurons.

Retraction Notice to: Mitochondrial Dynamics, Biogenesis, and Function Are Coordinated with the Cell Cycle by APC/CCDH1

(Cell Metabolism 15, 466–479; April 5, 2012)We have recently identified errors affecting several figure panels in Figures 4, 5, and S4, in which control data were processed inappropriately such that the figure panels do not accurately report the original data. While the conclusions reached in this paper may be sound, given the circumstances, the most responsible course of action is to retract the paper. A.G. regrets the inappropriate figure manipulations, of which his coauthors were completely unaware. We sincerely apologize to the scientific community for any confusion or adverse consequences resulting from the publication of these data.