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1.
STAR Protoc ; 4(4): 102717, 2023 Dec 15.
Article in English | MEDLINE | ID: mdl-37967016

ABSTRACT

Sensory neurons play pervasive roles throughout biology. In vitro studies to probe their functions hinge on the successful application of primary cell culture. Here, we present a protocol for the isolation and culture of mouse dorsal root ganglion neurons for imaging applications. We describe steps for extracting dorsal root ganglia, preparing cultures, maintaining them for days in vitro, and performing immunocytochemical labeling. We also include special considerations with respect to additional downstream applications. For complete details on the use and execution of this protocol, please refer to Smith et al. (2021).1.


Subject(s)
Ganglia, Spinal , Neurons , Mice , Animals , Neurons/physiology
2.
Curr Opin Struct Biol ; 77: 102484, 2022 Dec.
Article in English | MEDLINE | ID: mdl-36323134

ABSTRACT

All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes.


Subject(s)
Electron Microscope Tomography , Single Molecule Imaging , Cryoelectron Microscopy/methods , Electron Microscope Tomography/methods , Macromolecular Substances/chemistry
3.
Elife ; 112022 08 25.
Article in English | MEDLINE | ID: mdl-36005291

ABSTRACT

Previously, we showed that high-resolution template matching can localize ribosomes in two-dimensional electron cryo-microscopy (cryo-EM) images of untilted Mycoplasma pneumoniae cells with high precision (Lucas et al., 2021). Here, we show that comparing the signal-to-noise ratio (SNR) observed with 2DTM using different templates relative to the same cellular target can correct for local variation in noise and differentiate related complexes in focused ion beam (FIB)-milled cell sections. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. 2DTM can distinguish related molecular populations without the need to generate 3D reconstructions from the data to be classified, permitting classification even when only a few target particles exist in a cell.


Subject(s)
Saccharomyces cerevisiae Proteins , Cryoelectron Microscopy/methods , Likelihood Functions , Models, Molecular , Ribosomes , Saccharomyces cerevisiae
4.
J Biol Chem ; 298(8): 102224, 2022 08.
Article in English | MEDLINE | ID: mdl-35780835

ABSTRACT

During spliceosome assembly, the 3' splice site is recognized by sequential U2AF2 complexes, first with Splicing Factor 1 (SF1) and second by the SF3B1 subunit of the U2 small nuclear ribonuclear protein particle. The U2AF2-SF1 interface is well characterized, comprising a U2AF homology motif (UHM) of U2AF2 bound to a U2AF ligand motif (ULM) of SF1. However, the structure of the U2AF2-SF3B1 interface and its importance for pre-mRNA splicing are unknown. To address this knowledge gap, we determined the crystal structure of the U2AF2 UHM bound to a SF3B1 ULM site at 1.8-Å resolution. We discovered a distinctive trajectory of the SF3B1 ULM across the U2AF2 UHM surface, which differs from prior UHM/ULM structures and is expected to modulate the orientations of the full-length proteins. We established that the binding affinity of the U2AF2 UHM for the cocrystallized SF3B1 ULM rivals that of a nearly full-length U2AF2 protein for an N-terminal SF3B1 region. An additional SF3B6 subunit had no detectable effect on the U2AF2-SF3B1 binding affinities. We further showed that key residues at the U2AF2 UHM-SF3B1 ULM interface contribute to coimmunoprecipitation of the splicing factors. Moreover, disrupting the U2AF2-SF3B1 interface changed splicing of representative human transcripts. From analysis of genome-wide data, we found that many of the splice sites coregulated by U2AF2 and SF3B1 differ from those coregulated by U2AF2 and SF1. Taken together, these findings support distinct structural and functional roles for the U2AF2-SF1 and U2AF2-SF3B1 complexes during the pre-mRNA splicing process.


Subject(s)
RNA Precursors , RNA Splicing Factors/chemistry , RNA Splicing , Splicing Factor U2AF/chemistry , Humans , Ligands , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Binding , RNA Precursors/metabolism , RNA Splicing Factors/metabolism , Splicing Factor U2AF/metabolism
5.
Trends Biochem Sci ; 47(6): 477-491, 2022 06.
Article in English | MEDLINE | ID: mdl-35246374

ABSTRACT

In addition to their central functions in translation, ribosomes can adopt inactive structures that are fully assembled yet devoid of mRNA. We describe how the abundance of idle eukaryotic ribosomes is influenced by a broad range of biological conditions spanning viral infection, nutrient deprivation, and developmental cues. Vacant ribosomes may provide a means to exclude ribosomes from translation while also shielding them from degradation, and the variable identity of factors that occlude ribosomes may impart distinct functionality. We propose that regulated changes in the balance of idle and active ribosomes provides a means to fine-tune translation. We provide an overview of idle ribosomes, describe what is known regarding their function, and highlight questions that may clarify their biological roles.


Subject(s)
Ribosomal Proteins , Ribosomes , Polyribosomes/metabolism , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , Ribosomal Proteins/metabolism , Ribosomes/metabolism
6.
Viruses ; 14(2)2022 01 18.
Article in English | MEDLINE | ID: mdl-35215770

ABSTRACT

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use -1 programmed ribosomal frameshifting (-1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates -1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of -1 PRF in SARS-CoV-2 also inhibited -1 PRF in a range of bat CoVs-the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on -1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed -1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit -1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus/drug effects , Coronavirus/genetics , Frameshift Mutation , Frameshifting, Ribosomal/drug effects , Viral Proteins/antagonists & inhibitors , Animals , Antiviral Agents/therapeutic use , Chiroptera/virology , Coronavirus/classification , Coronavirus Infections/drug therapy , Nucleic Acid Conformation , RNA, Messenger/genetics , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , Viral Proteins/genetics , Virus Replication/drug effects
7.
Nat Commun ; 12(1): 6789, 2021 11 23.
Article in English | MEDLINE | ID: mdl-34815424

ABSTRACT

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.


Subject(s)
Elongation Factor 2 Kinase/metabolism , Peptide Elongation Factor 2/metabolism , Processing Bodies/metabolism , Ribosomes/metabolism , Animals , Cell Line, Tumor , Cryoelectron Microscopy , Elongation Factor 2 Kinase/genetics , Ganglia, Spinal/cytology , Humans , Male , Mice , Mice, Knockout , Nelfinavir/pharmacology , Phosphorylation/drug effects , Primary Cell Culture , Protein Biosynthesis/drug effects , Protein Biosynthesis/physiology , Sensory Receptor Cells/metabolism , Sensory Receptor Cells/ultrastructure
8.
J Neurosci ; 41(37): 7712-7726, 2021 09 15.
Article in English | MEDLINE | ID: mdl-34326146

ABSTRACT

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.


Subject(s)
Cytoskeletal Proteins/metabolism , Ganglia, Spinal/metabolism , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Signal Transduction/physiology , Vasodilation/physiology , Animals , Cytoskeletal Proteins/genetics , Inflammation/genetics , Inflammation/metabolism , Inflammation/physiopathology , Male , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Nociception/physiology , Nociceptors/physiology , Peripheral Nervous System Diseases/genetics , Peripheral Nervous System Diseases/metabolism , Peripheral Nervous System Diseases/physiopathology
9.
Elife ; 92020 05 19.
Article in English | MEDLINE | ID: mdl-32427100

ABSTRACT

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.


Subject(s)
Nucleic Acid Conformation , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribosomes , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cryoelectron Microscopy , DNA Polymerase III/genetics , DNA Polymerase III/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Frameshifting, Ribosomal , Fusion Proteins, gag-pol , Gene Expression Regulation, Bacterial , Gene Expression Regulation, Viral , HIV-1/genetics , HIV-1/metabolism , RNA, Bacterial , RNA, Messenger/chemistry , RNA, Transfer/chemistry
10.
Nat Commun ; 10(1): 1104, 2019 03 07.
Article in English | MEDLINE | ID: mdl-30846696

ABSTRACT

Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The obtained resolutions are 3.0 Å and 2.7 Å for the murine and human fibril, respectively. The two fibrils differ in fundamental properties, such as presence of right-hand or left-hand twisted cross-ß sheets and overall fold of the fibril proteins. Yet, both proteins adopt highly similar ß-arch conformations within the N-terminal ~21 residues. Our data demonstrate the importance of the fibril protein N-terminus for the stability of the analyzed amyloid fibril morphologies and suggest strategies of combating this disease by interfering with specific fibril polymorphs.


Subject(s)
Amyloid/metabolism , Amyloid/ultrastructure , Amyloidosis/metabolism , Amyloidosis/pathology , Amino Acid Sequence , Amyloid/genetics , Amyloidosis/genetics , Animals , Cryoelectron Microscopy , Female , Humans , Mice , Microscopy, Electron, Transmission , Middle Aged , Models, Molecular , Protein Conformation , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Stability , Sequence Homology, Amino Acid , Serum Amyloid A Protein/genetics , Serum Amyloid A Protein/metabolism , Serum Amyloid A Protein/ultrastructure , Species Specificity
11.
J Biol Chem ; 294(8): 2892-2902, 2019 02 22.
Article in English | MEDLINE | ID: mdl-30567737

ABSTRACT

The transcription elongation and pre-mRNA splicing factor Tat-SF1 associates with the U2 small nuclear ribonucleoprotein (snRNP) of the spliceosome. However, the direct binding partner and underlying interactions mediating the Tat-SF1-U2 snRNP association remain unknown. Here, we identified SF3b1 as a Tat-SF1-interacting subunit of the U2 snRNP. Our 1.1 Å resolution crystal structure revealed that Tat-SF1 contains a U2AF homology motif (UHM) protein-protein interaction module. We demonstrated that Tat-SF1 preferentially and directly binds the SF3b1 subunit compared with other U2AF ligand motif (ULM)-containing splicing factors, and further established that SF3b1 association depends on the integrity of the Tat-SF1 UHM. We next compared the Tat-SF1-binding affinities for each of the five known SF3b1 ULMs and then determined the structures of representative high- and low-affinity SF3b1 ULM complexes with the Tat-SF1 UHM at 1.9 Å and 2.1 Å resolutions, respectively. These structures revealed a canonical UHM-ULM interface, comprising a Tat-SF1 binding pocket for a ULM tryptophan (SF3b1 Trp338) and electrostatic interactions with a basic ULM tail. Importantly, we found that SF3b1 regulates Tat-SF1 levels and that these two factors influence expression of overlapping representative transcripts, consistent with a functional partnership of Tat-SF1 and SF3b1. Altogether, these results define a new molecular interface of the Tat-SF1-U2 snRNP complex for gene regulation.


Subject(s)
Phosphoproteins/metabolism , RNA Precursors/metabolism , RNA Splicing Factors/metabolism , RNA Splicing , Spliceosomes/metabolism , Splicing Factor U2AF/metabolism , Trans-Activators/metabolism , Amino Acid Sequence , Cell Nucleus/genetics , Cell Nucleus/metabolism , Crystallography, X-Ray , HEK293 Cells , Humans , Ligands , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Binding , Protein Conformation , Protein Interaction Domains and Motifs , RNA Precursors/genetics , RNA Splicing Factors/chemistry , RNA Splicing Factors/genetics , Sequence Homology , Spliceosomes/genetics , Splicing Factor U2AF/chemistry , Splicing Factor U2AF/genetics , Trans-Activators/chemistry , Trans-Activators/genetics
12.
Biophys J ; 111(12): 2570-2586, 2016 Dec 20.
Article in English | MEDLINE | ID: mdl-28002734

ABSTRACT

Splicing factor 1 (SF1) recognizes 3' splice sites of the major class of introns as a ternary complex with U2AF65 and U2AF35 splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF65 protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF65 binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3' splice site recognition by the SF1-U2AF65 complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF65 and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells.


Subject(s)
RNA Splice Sites , RNA Splicing Factors/chemistry , RNA Splicing Factors/metabolism , Amino Acid Sequence , Animals , Arginine/metabolism , Base Sequence , Humans , Models, Molecular , Mutation , Phosphorylation , Protein Domains , RNA Splice Sites/genetics , RNA Splicing Factors/genetics
13.
RNA ; 22(12): 1795-1807, 2016 12.
Article in English | MEDLINE | ID: mdl-27852923

ABSTRACT

U2AF homology motifs (UHM) that recognize U2AF ligand motifs (ULM) are an emerging family of protein-protein interaction modules. UHM-ULM interactions recur in pre-mRNA splicing factors including U2AF1 and SF3b1, which are frequently mutated in myelodysplastic syndromes. The core topology of the UHM resembles an RNA recognition motif and is often mistakenly classified within this large family. Here, we unmask the charade and review recent discoveries of UHM-ULM modules for protein-protein interactions. Diverse polypeptide extensions and selective phosphorylation of UHM and ULM family members offer new molecular mechanisms for the assembly of specific partners in the early-stage spliceosome.


Subject(s)
Proteins/chemistry , Ribonucleoproteins/metabolism , Humans , Ligands , Protein Binding
15.
J Biol Chem ; 289(25): 17325-37, 2014 Jun 20.
Article in English | MEDLINE | ID: mdl-24795046

ABSTRACT

U2AF homology motifs (UHMs) mediate protein-protein interactions with U2AF ligand motifs (ULMs) of pre-mRNA splicing factors. The UHM-containing alternative splicing factor CAPERα regulates splicing of tumor-promoting VEGF isoforms, yet the molecular target of the CAPERα UHM is unknown. Here we present structures of the CAPERα UHM bound to a representative SF3b155 ULM at 1.7 Å resolution and, for comparison, in the absence of ligand at 2.2 Å resolution. The prototypical UHM/ULM interactions authenticate CAPERα as a bona fide member of the UHM family of proteins. We identify SF3b155 as the relevant ULM-containing partner of full-length CAPERα in human cell extracts. Isothermal titration calorimetry comparisons of the purified CAPERα UHM binding known ULM-containing proteins demonstrate that high affinity interactions depend on the presence of an intact, intrinsically unstructured SF3b155 domain containing seven ULM-like motifs. The interplay among bound CAPERα molecules gives rise to the appearance of two high affinity sites in the SF3b155 ULM-containing domain. In conjunction with the previously identified, UHM/ULM-mediated complexes of U2AF(65) and SPF45 with SF3b155, this work demonstrates the capacity of SF3b155 to offer a platform for coordinated recruitment of UHM-containing splicing factors.


Subject(s)
Neoplasm Proteins/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Ribonucleoprotein, U2 Small Nuclear/metabolism , Amino Acid Motifs , HEK293 Cells , Humans , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Phosphoproteins/chemistry , Phosphoproteins/genetics , Protein Binding , Protein Structure, Quaternary , Protein Structure, Tertiary , RNA Splicing Factors , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Ribonucleoprotein, U2 Small Nuclear/chemistry , Ribonucleoprotein, U2 Small Nuclear/genetics
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