Project description:Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome. Fragile X syndrome is the most common form of inherited mental retardation affecting approximately 1 female out of 7000 and 1 male out of 4000 worldwide. The syndrome is due to the silencing of a single gene, the Fragile Mental Retardation 1 (FMR1), that codes for the Fragile X mental retardation protein (FMRP). This protein is highly expressed in brain and controls local protein synthesis essential for neuronal development and maturation as well as the formation of neural circuits. Several studies suggest a role for FMRP in the regulation of mRNA transport along axons and dendrites to distant synaptic locations in structures called RNA granules. Here we report the isolation of a particular subpopulation of these structures and the analysis of their architecture and composition in terms of RNA and protein. Also, using time-lapse video microscopy, we monitored granule transport and fusion throughout neuronal processes. These findings open new avenues for the study of RNA transport dysfunctions in animal models of nervous system disorders. The control or reference structure or subcellular fraction are the neuronal polyribosomes while the experimental or test structure or subcellular fraction are the neuronal granules. Three independant replicates were done for each structure. Microarray hybridization was done using a two-color design in full dye-swap.

Project description:It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from non-specific interactors, preserving and isolating all unique interactions including those that are weak, transient or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact sub-micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under non-denaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry (SAC-MS). Here, we demonstrate that SAC-MS allows us to stabilize and elucidate local, distant and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.

Project description:Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome. Fragile X syndrome is the most common form of inherited mental retardation affecting approximately 1 female out of 7000 and 1 male out of 4000 worldwide. The syndrome is due to the silencing of a single gene, the Fragile Mental Retardation 1 (FMR1), that codes for the Fragile X mental retardation protein (FMRP). This protein is highly expressed in brain and controls local protein synthesis essential for neuronal development and maturation as well as the formation of neural circuits. Several studies suggest a role for FMRP in the regulation of mRNA transport along axons and dendrites to distant synaptic locations in structures called RNA granules. Here we report the isolation of a particular subpopulation of these structures and the analysis of their architecture and composition in terms of RNA and protein. Also, using time-lapse video microscopy, we monitored granule transport and fusion throughout neuronal processes. These findings open new avenues for the study of RNA transport dysfunctions in animal models of nervous system disorders.

			Action	DRS
	SRR8080090.fastq.gz	Fastqsanger.gz
	SRR8080100.fastq.gz	Fastqsanger.gz
	SRR8080110.fastq.gz	Fastqsanger.gz
	SRR8080120.fastq.gz	Fastqsanger.gz
	SRR8080130.fastq.gz	Fastqsanger.gz

Dataset Information

Affinity capture of polyribosomes followed by RNAseq (ACAPseq), a discovery platform for protein-protein interactions

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