Project description:In neurons, mRNAs and associated RNA-binding proteins assemble into ribonucleoprotein (RNP) granules essential to regulate mRNA trafficking, local translation, and turnover. Dysregulation of RNA-protein condensation can disturb synaptic plasticity. We report that the novel lncRNA mimi is a constitutive and essential component of large cytoplasmic condensates (RNP granules) in fly neurons that are biochemically enriched by differential centrifugation. Here, employing relative iBAQ quantification we carry out a differential proteomic analysis of cytoplasmic RNP granules in wild-type versus delta-mimi mutant fly brains. Brain lysates from wild-type and mutant flies serve as a general proteome input control.
Project description:Extracellular proteins play key roles in microbial biofilm and aggregate formation, yet remain poorly understood. Using two advanced metaproteomic techniques, this study identified and classified 387 extracellular proteins potentially involved in aggregate formation, including filamentous and cell surface proteins from various organisms. The findings suggest that diverse proteins from multiple organisms support the granular biofilm formation in the investigated Ca. Accumulibacter enrichment, providing a valuable resource for studying the scope of extracellular proteins.
Project description:Circular RNAs (circRNAs) are covalently closed single-stranded RNA molecules produced by reverse splicing of the precursor mRNAs of thousands of gene exons in eukaryotes. Some circRNAs play potentially important roles at the molecular level through different modes of action in physiological and pathological conditions. In recent years, it has been found that differentially expressed circRNAs are present in chondrocyte degeneration in OA and are able to modulate the inflammatory response in chondrocyte degeneration. However, the molecular mechanisms of how circRNAs regulate the inflammatory response during chondrogenic degeneration in OA have not been adequately studied. Are there other circRNAs involved in regulating the inflammatory response during knee OA chondrocyte degeneration and are there other molecular mechanisms by which circRNAs regulate chondrocyte degeneration? These scientific questions deserve relevant studies.
Project description:Eukaryotic cells have to prevent the export of unspliced pre-mRNAs until intron removal is completed to avoid the expression of aberrant and potentially harmful proteins. Only mature RNAs associate with the export receptor Mex67 (mammalian TAP) and enter the cytoplasm. The underlying nuclear quality control mechanisms are still unclear. Here we show that two shuttling SR-proteins Gbp2 and Hrb1 are key surveillance factors for the selective export of spliced mRNAs in yeast. Their absence leads to the significant leakage of unspliced pre-mRNAs into the cytoplasm. They bind to pre-mRNAs and the spliceosome during splicing, where they are necessary for the surveillance of splicing and the stable binding of the TRAMP-complex to the spliceosome-bound transcripts. Faulty transcripts are marked for their degradation at the nuclear exosome. On correct mRNAs the SR-proteins recruit Mex67 upon completion of splicing to allow a quality controlled nuclear export. Altogether, these data identify a role for shuttling SR-proteins in mRNA surveillance and nuclear mRNA quality control. 6 samples, i.e. 2 replicates per protein Gbp2, Hrb1 and Npl3
Project description:Stress granules (SGs) and processing bodies (PBs) are membraneless cytoplasmic assemblies regulating mRNAs under environmental stress such as viral infections, neurological disorders, or cancer. Upon antigen stimulation, T lymphocytes mediate their immune functions under regulatory mechanisms involving SGs and PBs. However, the impact of T cell activation on such complexes, in term of formation, constitution and relationship remains unknown. Here, by combining proteomic, transcriptomic and immunofluorescence approaches, we simultaneously characterized the SGs and PBs from primary human T lymphocytes pre- and post-stimulation. The proteomes and transcriptomes of SGs and PBs were identified, unveiling an unanticipated molecular and functional complementarity. Notwithstanding, these granules keep distinct spatial organizations and abilities to interact with mRNAs. This comprehensive characterization of the RNP granule proteomic and transcriptomic landscapes provides a unique resource for future investigations on SGs and PBs in T lymphocytes.
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:This dataset was utilized to assess the performance of a novel de novo metaproteomics pipeline, which performs sequence alignment of de novo sequences from complete metaproteomics experiments. Traditionally, metaproteomics data annotation relies on database searching that requires sample-specific databases derived from whole metagenome sequencing experiments. Creating these databases, however, is a complex, time-consuming, and error prone process, which can introduce biases affecting the outcomes and conclusions, highlighting the need for alternative methods. The evaluated approach offers rapid and orthogonal insights into metaproteomics data.
Project description:Biogenesis of inclusion bodies (IBs) facilitates protein quality control (PQC). Canonical aggresomes execute degradation of misfolded proteins while non-degradable amyloids quarantine into Insoluble Protein Deposits. Lewy Bodies (LBs) are well-known neurodegenerative IBs of α-Synuclein but PQC-benefits and drawbacks associated with LBs remain underexplored. Here, we report that a crosstalk between LBs and aggresome-like IBs of α-Synuclein (Syn-aggresomes) buffer amyloidogenic α-Synuclein load. LBs possess unorthodox PQC-capacities of self-quarantining Syn-amyloids and being degradable upon receding fresh amyloidogenesis. Syn-aggresomes equilibrate biogenesis of LBs by facilitating spontaneous degradation of soluble α-Synuclein and opportunistic turnover of Syn-amyloids. LBs overgrow at the perinucleus once amyloidogenesis sets in and are misidentified by cytosolic BICD2 as cargos for motor-protein dynein. Simultaneously, microtubules surrounding the perinuclear LBs are distorted misbalancing the dynein motor-force on nucleoskeleton leading to widespread lamina injuries. Like typical Laminopathies, nucleocytoplasmic mixing, DNA-damage, and deregulated transcription of stress chaperones defeat the proteostatic purposes of LBs.
Project description:Stress granules are large messenger ribonucleoprotein (mRNP) aggregates composed of translation initiation factors and mRNAs that appear when the cell encounters various stressors. Current dogma indicates that stress granules function as inert storage depots for translationally silenced mRNPs until the cell signals for renewed translation and stress granule disassembly. We used RasGAP SH3-binding protein (G3BP) overexpression to induce stress granules and study their assembly process and signaling to the translation apparatus. We found that assembly of large G3BP-induced stress granules, but not small granules, precedes phosphorylation of eIF2?. Using mouse embryonic fibroblasts depleted for individual eukaryotic initiation factor 2? (eIF2?) kinases, we identified protein kinase R as the principal kinase that mediates eIF2? phosphorylation by large G3BP-induced granules. These data indicate that increasing stress granule size is associated with a threshold or switch that must be triggered in order for eIF2? phosphorylation and subsequent translational repression to occur. Furthermore, these data suggest that stress granules are active in signaling to the translational machinery and may be important regulators of the innate immune response.