-
Science (New York, N.Y.) Jun 2022INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in...
INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Y‑shaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartment‑specific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for disease‑associated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cell‑based assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiled‑coil hub that tethers two separate mRNP‑remodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoan‑specific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an N‑terminal S‑shaped α‑helical solenoid followed by a coiled‑coil oligomerization element, numerous Ran‑interacting domains, an E3 ligase domain, and a C‑terminal prolyl‑isomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their N‑terminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cell‑based assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryo‑ET density matched the dimensions of the CFNC coiled‑coil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiled‑coil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryo‑ET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our near‑atomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].
Topics: Chaetomium; Cryoelectron Microscopy; Cytoplasm; Fungal Proteins; Humans; Molecular Chaperones; Nuclear Pore; Nuclear Pore Complex Proteins; Protein Conformation; RNA Transport; RNA, Messenger
PubMed: 35679405
DOI: 10.1126/science.abm9129 -
Developmental Neurobiology Mar 2014Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in cell body responses to axotomy. Recent studies have begun... (Review)
Review
Axonal protein synthesis has been shown to play a role in developmental and regenerative growth, as well as in cell body responses to axotomy. Recent studies have begun to identify the protein products that contribute to these autonomous responses of axons. In the peripheral nervous system, intra-axonal protein synthesis has been implicated in the localized in vivo responses to neuropathic stimuli, and there is emerging evidence for protein synthesis in CNS axons in vivo. Despite that hundreds of mRNAs have now been shown to localize into the axonal compartment, knowledge of what RNA binding proteins are responsible for this is quite limited. Here, we review the current state of knowledge of RNA transport mechanisms and highlight recently uncovered mechanisms for dynamically altering the axonal transcriptome. Both changes in the levels or activities of components of the RNA transport apparatus and alterations in transcription of transported mRNAs can effectively shift the axonal mRNA population. Consistent with this, the axonal RNA population shifts with development, with changes in growth state, and in response to extracellular stimulation. Each of these events must impact the transcriptional and transport apparatuses of the neuron, thus directly and indirectly modifying the axonal transcriptome.
Topics: Animals; Axons; Humans; Nervous System Diseases; RNA Transport; RNA, Messenger; RNA-Binding Proteins; Transcriptome
PubMed: 23959706
DOI: 10.1002/dneu.22123 -
Biochimica Et Biophysica Acta 2012The mitochondrial genome encodes a very small fraction of the macromolecular components that are required to generate functional mitochondria. Therefore, most components... (Review)
Review
The mitochondrial genome encodes a very small fraction of the macromolecular components that are required to generate functional mitochondria. Therefore, most components are encoded within the nuclear genome and are imported into mitochondria from the cytosol. Understanding how mitochondria are assembled, function, and dysfunction in diseases requires detailed knowledge of mitochondrial import mechanisms and pathways. The import of nucleus-encoded RNAs is required for mitochondrial biogenesis and function, but unlike pre-protein import, the pathways and cellular machineries of RNA import are poorly defined, especially in mammals. Recent studies have shown that mammalian polynucleotide phosphorylase (PNPASE) localizes in the mitochondrial intermembrane space (IMS) to regulate the import of RNA. The identification of PNPASE as the first component of the RNA import pathway, along with a growing list of nucleus-encoded RNAs that are imported and newly developed assay systems for RNA import studies, suggest a unique opportunity is emerging to identify the factors and mechanisms that regulate RNA import into mammalian mitochondria. Here we summarize what is known in this fascinating area of mitochondrial biogenesis, identify areas that require further investigation, and speculate on the impact unraveling RNA import mechanisms and pathways will have for the field going forward. This article is part of a Special Issue entitled: Mitochondrial Gene Expression.
Topics: Animals; Cell Nucleus; Exoribonucleases; Humans; Mitochondria; Oxidative Phosphorylation; Plants; RNA Transport; RNA, Nuclear; RNA, Transfer; Saccharomyces cerevisiae
PubMed: 22023881
DOI: 10.1016/j.bbagrm.2011.10.001 -
RNA Biology Feb 2021La and La-related proteins (LARPs) are characterized by a common RNA interaction platform termed the La module. This structural hallmark allows LARPs to pervade various... (Review)
Review
La and La-related proteins (LARPs) are characterized by a common RNA interaction platform termed the La module. This structural hallmark allows LARPs to pervade various aspects of RNA biology. The metazoan LARP7 protein binds to the 7SK RNA as part of a 7SK small nuclear ribonucleoprotein (7SK snRNP), which inhibits the transcriptional activity of RNA polymerase II (Pol II). Additionally, recent findings revealed unanticipated roles of LARP7 in the assembly of other RNPs, as well as in the modification, processing and cellular transport of RNA molecules. Reduced levels of functional LARP7 have been linked to cancer and Alazami syndrome, two seemingly unrelated human diseases characterized either by hyperproliferation or growth retardation. Here, we review the intricate regulatory networks centered on LARP7 and assess how malfunction of these networks may relate to the etiology of LARP7-linked diseases.
Topics: Humans; Nucleic Acid Conformation; RNA Processing, Post-Transcriptional; RNA Stability; RNA Transport; RNA, Untranslated; RNA-Binding Proteins; Ribonucleoproteins
PubMed: 32401147
DOI: 10.1080/15476286.2020.1767952 -
RNA Biology Mar 2021As the adaptor that decodes mRNA sequence into protein, the basic aspects of tRNA structure and function are central to all studies of biology. Yet the complexities of... (Review)
Review
As the adaptor that decodes mRNA sequence into protein, the basic aspects of tRNA structure and function are central to all studies of biology. Yet the complexities of their properties and cellular roles go beyond the view of tRNAs as static participants in protein synthesis. Detailed analyses through more than 60 years of study have revealed tRNAs to be a fascinatingly diverse group of molecules in form and function, impacting cell biology, physiology, disease and synthetic biology. This review analyzes tRNA structure, biosynthesis and function, and includes topics that demonstrate their diversity and growing importance.
Topics: Animals; Evolution, Molecular; Gene Expression Regulation; Genetic Code; Humans; Nucleic Acid Conformation; Protein Biosynthesis; RNA Folding; RNA Processing, Post-Transcriptional; RNA Splicing; RNA Transport; RNA, Transfer; Ribosomes; Structure-Activity Relationship; Transcription, Genetic; Transfer RNA Aminoacylation
PubMed: 32900285
DOI: 10.1080/15476286.2020.1809197 -
Nature Communications Sep 2023PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved...
PGC-1α plays a central role in maintaining mitochondrial and energy metabolism homeostasis, linking external stimuli to transcriptional co-activation of genes involved in adaptive and age-related pathways. The carboxyl-terminus encodes a serine/arginine-rich (RS) region and an RNA recognition motif, however the RNA-processing function(s) were poorly investigated over the past 20 years. Here, we show that the RS domain of human PGC-1α directly interacts with RNA and the nuclear RNA export receptor NXF1. Inducible depletion of PGC-1α and expression of RNAi-resistant RS-deleted PGC-1α further demonstrate that its RNA/NXF1-binding activity is required for the nuclear export of some canonical mitochondrial-related mRNAs and mitochondrial homeostasis. Genome-wide investigations reveal that the nuclear export function is not strictly linked to promoter-binding, identifying in turn novel regulatory targets of PGC-1α in non-homologous end-joining and nucleocytoplasmic transport. These findings provide new directions to further elucidate the roles of PGC-1α in gene expression, metabolic disorders, aging and neurodegeneration.
Topics: Humans; Active Transport, Cell Nucleus; Gene Expression; Homeostasis; RNA; RNA Transport
PubMed: 37679383
DOI: 10.1038/s41467-023-41304-8 -
Journal of Biosciences Dec 2019Translin, a highly conserved, DNA/RNA binding protein, is abundantly expressed in brain, testis and in certain malignancies. It was discovered initially in the quest to... (Review)
Review
Translin, a highly conserved, DNA/RNA binding protein, is abundantly expressed in brain, testis and in certain malignancies. It was discovered initially in the quest to find proteins that bind to alternating polypurines-polypyrimidines repeats. It has been implicated to have a role in RNA metabolism (tRNA processing, RNAi, RNA transport, etc.), transcription, DNA damage response, etc. Studies from human, mice, drosophila and yeast have revealed that it forms an octameric ring, which is important for its function. Translin is a cytoplasmic protein, but under genotoxic stress, it migrates into the nucleus, binds to the break point hot spots and therefore, thought to be involved in chromosomal translocation events as well as DNA damage related response. Its structure is known and DNA binding regions, GTP binding region and regions responsible for homotypic and heterotypic interaction are known. It forms a ball like structure with open central channel for accommodating the substrate nucleic acids. Besides this, translin protein binds to 3' and 5' UTR of certain mRNAs and probably regulates their availability for translation. It is also involved in mRNA transport and cell cycle progression. It forms a heteromeric complex with translin associated factor-X (TRAX) to form C3PO complex which is involved in RNA silencing process. Recently, it has been shown that translin is upregulated under starvation conditions in Drosophila and is involved in the integration of sleep and metabolic rate of the flies. Earlier studies classified translin as a DNA repair protein; however subsequent studies showed that it is a multifunctional protein. With this background, in this review we have summarized the translin biochemical activities, cellular function as well as structural properties of this important protein.
Topics: Animals; DNA Repair; DNA-Binding Proteins; Drosophila Proteins; Humans; Mice; Nucleic Acids; RNA Transport; RNA, Messenger; RNA-Binding Proteins
PubMed: 31894120
DOI: No ID Found -
Molecules and Cells Dec 2016Local protein synthesis mediates precise spatio-temporal regulation of gene expression for neuronal functions such as long-term plasticity, axon guidance and... (Review)
Review
Local protein synthesis mediates precise spatio-temporal regulation of gene expression for neuronal functions such as long-term plasticity, axon guidance and regeneration. To reveal the underlying mechanisms of local translation, it is crucial to understand mRNA transport, localization and translation in live neurons. Among various techniques for mRNA analysis, fluorescence microscopy has been widely used as the most direct method to study localization of mRNA. Live-cell imaging of single RNA molecules is particularly advantageous to dissect the highly heterogeneous and dynamic nature of messenger ribonucleoprotein (mRNP) complexes in neurons. Here, we review recent advances in the study of mRNA localization and translation in live neurons using novel techniques for single-RNA imaging.
Topics: Animals; Humans; Neurons; Protein Biosynthesis; RNA Transport; RNA, Messenger
PubMed: 28030897
DOI: 10.14348/molcells.2016.0277 -
Open Biology Sep 2020Messenger RNA (mRNA) localization allows spatiotemporal regulation of the proteome at the subcellular level. This is observed in the axons of neurons, where mRNA... (Review)
Review
Messenger RNA (mRNA) localization allows spatiotemporal regulation of the proteome at the subcellular level. This is observed in the axons of neurons, where mRNA localization is involved in regulating neuronal development and function by orchestrating rapid adaptive responses to extracellular cues and the maintenance of axonal homeostasis through local translation. Here, we provide an overview of the key findings that have broadened our knowledge regarding how specific mRNAs are trafficked and localize to axons. In particular, we review transcriptomic studies investigating mRNA content in axons and the molecular principles underpinning how these mRNAs arrived there, including cis-acting mRNA sequences and trans-acting proteins playing a role. Further, we discuss evidence that links defective axonal mRNA localization and pathological outcomes.
Topics: Animals; Axons; Binding Sites; Gene Expression Profiling; Gene Expression Regulation; Humans; Neurons; Protein Transport; RNA Transport; RNA, Messenger; RNA-Binding Proteins; Response Elements; Transcriptome
PubMed: 32961072
DOI: 10.1098/rsob.200177 -
The EMBO Journal Aug 2011In most eukaryotes, double-stranded RNA is processed into small RNAs that are potent regulators of gene expression. This gene silencing process is known as RNA silencing... (Review)
Review
In most eukaryotes, double-stranded RNA is processed into small RNAs that are potent regulators of gene expression. This gene silencing process is known as RNA silencing or RNA interference (RNAi) and, in plants and nematodes, it is associated with the production of a mobile signal that can travel from cell-to-cell and over long distances. The sequence-specific nature of systemic RNA silencing indicates that a nucleic acid is a component of the signalling complex. Recent work has shed light on the mobile RNA species, the genes involved in the production and transport of the signal. This review discusses the advances in systemic RNAi and presents the current challenges and questions in this rapidly evolving field.
Topics: Animals; Gene Silencing; Humans; Mice; Nematoda; Plants; RNA Interference; RNA Transport; RNA, Double-Stranded; RNA, Plant; RNA, Small Interfering; Signal Transduction
PubMed: 21878996
DOI: 10.1038/emboj.2011.274