Biomolecular condensates amplify mRNA decapping by biasing enzyme conformation.
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ABSTRACT:
SUBMITTER: Tibble RW
PROVIDER: S-EPMC8476181 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Decapping of mRNA is a critical step in eukaryotic mRNA turnover, yet the proteins involved in this activity remain elusive in mammals. We identified the human Dcp2 protein (hDcp2) as an enzyme containing intrinsic decapping activity. hDcp2 specifically hydrolyzed methylated capped RNA to release m(7)GDP; however, it did not function on the cap structure alone. hDcp2 is therefore functionally distinct from the recently identified mammalian scavenger decapping enzyme, DcpS. hDcp2-mediated decapping required a functional Nudix (nucleotide diphosphate linked to an X moiety) pyrophosphatase motif as mutations in conserved amino acids within this motif disrupted the decapping activity. hDcp2 is detected exclusively in the cytoplasm and predominantly cosediments with polysomes. Consistent with the localization of hDcp2, endogenous Dcp2-like decapping activity was detected in polysomal fractions prepared from mammalian cells. Similar to decapping in yeast, the presence of the poly(A) tail was inhibitory to the endogenous decapping activity, yet unlike yeast, competition of cap-binding proteins by cap analog did not influence the efficiency of decapping. Therefore the mammalian homologue of the yeast Dcp2 protein is an mRNA decapping enzyme demonstrated to contain intrinsic decapping activity.
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Project description:Removal of the 5'-end 7-methylguanosine cap structure is a critical step in the highly regulated process of mRNA decay. The Nudix hydrolase, Dcp2, was identified as a first decapping enzyme and subsequently shown to preferentially modulate stability of only a subset of mRNAs. This observation led to the hypothesis that mammalian cells possess multiple decapping enzymes that may function in distinct pathways. Here we report Nudt3 is a Nudix protein that possesses mRNA decapping activity in cells and is a modulator of MCF-7 breast cancer cell migration. Reduction of Nudt3 protein levels in MCF-7 cells promotes increased cell migration and corresponding enhanced filopodia extensions. Importantly, this phenotype was reversed by complementation with wild type, but not catalytically inactive Nudt3 protein indicating Nudt3 decapping activity normally functions to control cell migration. Genome-wide analysis of Nudt3 compromised cells identified elevated levels of transcripts involved in cell motility including integrin β6, lipocalin-2, and fibronectin. The observed increase in mRNA abundance was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 knockdown cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude that Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals.
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Project description:Fyn is a Src kinase that controls critical signalling cascades and has been implicated in learning and memory. Postsynaptic enrichment of Fyn underpins synaptotoxicity in dementias such as Alzheimer's disease and frontotemporal lobar degeneration with Tau pathology (FTLD-Tau). The FLTD P301L mutant Tau is associated with a higher propensity to undergo liquid-liquid phase separation (LLPS) and form biomolecular condensates. Expression of P301L mutant Tau promotes aberrant trapping of Fyn in nanoclusters within hippocampal dendrites by an unknown mechanism. Here, we used single-particle tracking photoactivated localisation microscopy to demonstrate that the opening of Fyn into its primed conformation promotes its nanoclustering in dendrites leading to increased Fyn/ERK/S6 downstream signalling. Preventing the auto-inhibitory closed conformation of Fyn through phospho-inhibition or through perturbation of its SH3 domain increased Fyn's nanoscale trapping, whereas inhibition of the catalytic domain had no impact. By combining pharmacological and genetic approaches, we demonstrate that P301L Tau enhanced both Fyn nanoclustering and Fyn/ERK/S6 signalling via its ability to form biomolecular condensates. Together, our findings demonstrate that Fyn alternates between a closed and an open conformation, the latter being enzymatically active and clustered. Furthermore, pathogenic immobilisation of Fyn relies on the ability of P301L Tau to form biomolecular condensates, thus highlighting the critical importance of LLPS in controlling nanoclustering and downstream intracellular signalling events.
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Project description:Evidence accumulated over the past decade provides support for liquid-liquid phase separation as the mechanism underlying the formation of biomolecular condensates, which include not only 'membraneless' organelles such as nucleoli and RNA granules, but additional assemblies involved in transcription, translation and signaling. Understanding the molecular mechanisms of condensate function requires knowledge of the structures of their constituents. Current knowledge suggests that structures formed via multivalent domain-motif interactions remain largely unchanged within condensates. Two different viewpoints exist regarding structures of disordered low-complexity domains within condensates; one argues that low-complexity domains remain largely disordered in condensates and their multivalency is encoded in short motifs called 'stickers', while the other argues that the sequences form cross-? structures resembling amyloid fibrils. We review these viewpoints and highlight outstanding questions that will inform structure-function relationships for biomolecular condensates.
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Project description:Nuclear processes such as DNA replication, transcription, and RNA processing each depend on the concerted action of many different protein and RNA molecules. How biomolecules with shared functions find their way to specific locations has been assumed to occur largely by diffusion-mediated collisions. Recent studies have shown that many nuclear processes occur within condensates that compartmentalize and concentrate the protein and RNA molecules required for each process, typically at specific genomic loci. These condensates have common features and emergent properties that provide the cell with regulatory capabilities beyond canonical molecular regulatory mechanisms. We describe here the shared features of nuclear condensates, the components that produce locus-specific condensates, elements of specificity, and the emerging understanding of mechanisms regulating these compartments.
| S-EPMC7572565 | biostudies-literature
Project description:The Arabidopsis thaliana decapping enzyme (AtDcp2) was characterized by bioinformatics analysis and by biochemical studies of the enzyme and mutants produced by recombinant expression. Three functionally significant regions were detected: (i) a highly disordered C-terminal region with a putative PSD-95, Discs-large, ZO-1 (PDZ) domain-binding motif, (ii) a conserved Nudix box constituting the putative active site and (iii) a putative RNA binding domain consisting of the conserved Box B and a preceding loop region. Mutation of the putative PDZ domain-binding motif improved the stability of recombinant AtDcp2 and secondary mutants expressed in Escherichia coli. Such recombinant AtDcp2 specifically hydrolysed capped mRNA to produce 7-methyl GDP and decapped RNA. AtDcp2 activity was Mn(2+)- or Mg(2+)-dependent and was inhibited by the product 7-methyl GDP. Mutation of the conserved glutamate-154 and glutamate-158 in the Nudix box reduced AtDcp2 activity up to 400-fold and showed that AtDcp2 employs the catalytic mechanism conserved amongst Nudix hydrolases. Unlike many Nudix hydrolases, AtDcp2 is refractory to inhibition by fluoride ions. Decapping was dependent on binding to the mRNA moiety rather than to the 7-methyl diguanosine triphosphate cap of the substrate. Mutational analysis of the putative RNA-binding domain confirmed the functional significance of an 11-residue loop region and the conserved Box B.
| S-EPMC2248764 | biostudies-literature
Project description:Homozygous mutations in the gene encoding the scavenger mRNA-decapping enzyme, DcpS, have been shown to underlie developmental delay and intellectual disability. Intellectual disability is associated with both abnormal neocortical development and mRNA metabolism. However, the role of DcpS and its scavenger decapping activity in neuronal development is unknown. Here, we show that human neurons derived from patients with a DcpS mutation have compromised differentiation and neurite outgrowth. Moreover, in the developing mouse neocortex, DcpS is required for the radial migration, polarity, neurite outgrowth, and identity of developing glutamatergic neurons. Collectively, these findings demonstrate that the scavenger mRNA decapping activity contributes to multiple pivotal roles in neural development and further corroborate that mRNA metabolism and neocortical pathologies are associated with intellectual disability.
| S-EPMC8971079 | biostudies-literature
Project description:Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid-liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.
| S-EPMC7434221 | biostudies-literature
Project description:Biomolecular condensates are membraneless organelle-like structures that can concentrate molecules and often form through liquid-liquid phase separation. Biomolecular condensate assembly is tightly regulated by developmental and environmental cues. Although research on biomolecular condensates has intensified in the past 10 years, our current understanding of the molecular mechanisms and components underlying their formation remains in its infancy, especially in plants. However, recent studies have shown that the formation of biomolecular condensates may be central to plant acclimation to stress conditions. Here, we describe the mechanism, regulation, and properties of stress-related condensates in plants, focusing on stress granules and processing bodies, 2 of the most well-characterized biomolecular condensates. In this regard, we showcase the proteomes of stress granules and processing bodies in an attempt to suggest methods for elucidating the composition and function of biomolecular condensates. Finally, we discuss how biomolecular condensates modulate stress responses and how they might be used as targets for biotechnological efforts to improve stress tolerance.
| S-EPMC10473214 | biostudies-literature
Project description:The Dcp2 and Nudt16 Nudix hydrolases, are mRNA decapping enzymes that preferentially modulate stability of a subset of mRNAs. Here we report Nudt3 is a third Nudix protein that possess mRNA decapping activity in cells and is a modulator of cell migration in MCF-7 breast cancer cells. Genome-wide analysis of Nudt3 compromised cells identified increases in mRNAs involved in cell motility including integrin β6, lipocalin-2 and fibronectin. The increase in mRNA levels was dependent on Nudt3 decapping activity where integrin β6 and lipocalin-2 were modulated directly through mRNA stability, while fibronectin was indirectly controlled. Moreover, increased cell migration observed in Nudt3 depleted cells was mediated through the extracellular integrin β6 and fibronectin protein nexus. We conclude, Nudt3 is an mRNA decapping enzyme that orchestrates expression of a subset of mRNAs to modulate cell migration and further substantiates the existence of multiple decapping enzymes functioning in distinct cellular pathways in mammals. Stably transformed MCF-7 cell lines constitutively expressing either a short hairpin RNA (shRNA) directed against Nudt3 (Nudt3KD) or a non-targeted control shRNA (ConKD) were used, with three replicate cultures used per group (n=3).
2016-07-21 | E-GEOD-75146 | biostudies-arrayexpress