ABSTRACT
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.
Subject(s)
Fluorescent Dyes/chemistry , RNA, Messenger/metabolism , Single Molecule Imaging/methods , 3' Untranslated Regions , 5' Untranslated Regions , Cell Line, Tumor , Genes, Reporter , HEK293 Cells , Humans , Peptide Chain Initiation, Translational , RNA, Messenger/chemistry , Ribosomes/metabolism , Single-Domain Antibodies/chemistry , Single-Domain Antibodies/immunologyABSTRACT
Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.
Subject(s)
Poisons , Humans , Cold Shock Proteins and Peptides/metabolism , Cold Temperature , RNA, Messenger/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
Subject(s)
Codon, Nonsense/genetics , Nonsense Mediated mRNA Decay/genetics , RNA, Messenger/genetics , Codon, Nonsense/chemistry , Exons/genetics , Humans , Introns/genetics , Mutation/genetics , RNA Stability/genetics , RNA, Messenger/chemistry , Single Molecule ImagingABSTRACT
Small extracellular vesicle (sEV)-mediated intercellular communication regulates multiple aspects of growth and development in multicellular organisms. However, the mechanism underlying cargo recruitment into sEVs is currently unclear. We show that the key nucleo-cytoplasmic transport (NCT) protein-RanGTPase, in its GTP-bound form (RanGTP), is enriched in sEVs secreted by mammalian cells. This recruitment of RanGTP into sEVs depends on the export receptor CRM1 (also called XPO1). The recruitment of GAPDH, a candidate cargo protein, into sEVs is regulated by the RanGTP-CRM1axis in a nuclear export signal (NES)-dependent manner. Perturbation of NCT through overexpression or depletion of nuclear transport components affected the recruitment of Ran, CRM1 and GAPDH into sEVs. Our studies, thus, suggest a link between NCT, particularly the Ran-CRM1 axis, and recruitment of NES-containing cargoes into the sEVs. Collectively, these findings implicate RanGTPase as a link between NCT and sEV mediated intercellular communication.
Subject(s)
Cell Communication , Extracellular Vesicles , Active Transport, Cell Nucleus , Animals , Mammals , Nuclear Export SignalsABSTRACT
Fluorogenic RNA aptamers bind and activate the fluorescence of otherwise nonfluorescent dyes. However, fluorogenic aptamers are limited by the small number of fluorogenic dyes suitable for use in live cells. In this communication, fluorogenic proteins whose fluorescence is activated by RNA aptamers are described. Fluorogenic proteins are highly unstable until they bind RNA aptamers inserted into messenger RNAs, resulting in fluorescent RNA-protein complexes that enable live imaging of mRNA in living cells.
Subject(s)
Aptamers, Nucleotide/metabolism , Fluorescence , Fluorescent Dyes/chemistry , Microscopy, Fluorescence/methods , Molecular Imaging/methods , RNA, Messenger/analysis , Aptamers, Nucleotide/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , HEK293 Cells , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolismABSTRACT
MicroRNA (miRNA)-mediated translational suppression of mRNAs is involved in the regulation of multiple cellular processes. A recent study showed that Nup358, a protein mutated in a neurological disorder called acute necrotizing encephalopathy 1 (ANE1), helps in the coupling of miRNA-induced silencing complex (miRISC) - consisting of miRNA, AGO and GW182/TNRC6 proteins - with the target mRNA. Here we provide a detailed characterization of the interaction between Nup358 and GW182. We identified that the N-terminal region of Nup358 directly interacts with the C-terminal silencing domain of GW182. Interestingly, ANE1-associated Nup358 mutants display reduced interaction with GW182. Consistent with this, one of the prevalent ANE1 mutations, 585th threonine (T) residue changed to methionine (M) [T585M] compromised Nup358's ability to function in the miRNA pathway. Collectively, these results suggest that the ANE1-associated mutations in Nup358 might affect the miRNA pathway and contribute to the development of ANE1.
Subject(s)
Autoantigens/metabolism , Brain Diseases/genetics , MicroRNAs/metabolism , Molecular Chaperones/metabolism , Mutation/genetics , Nuclear Pore Complex Proteins/metabolism , RNA-Binding Proteins/metabolism , Animals , Autoantigens/chemistry , Cell Line , Humans , MicroRNAs/genetics , Protein Binding , Protein Domains , RNA-Binding Proteins/chemistryABSTRACT
MicroRNA (miRNA)-guided mRNA repression, mediated by the miRNA-induced silencing complex (miRISC), is an important component of post-transcriptional gene silencing. However, how miRISC identifies the target mRNA in vivo is not well understood. Here, we show that the nucleoporin Nup358 plays an important role in this process. Nup358 localizes to the nuclear pore complex and to the cytoplasmic annulate lamellae (AL), and these structures dynamically associate with two mRNP granules: processing bodies (P bodies) and stress granules (SGs). Nup358 depletion disrupts P bodies and concomitantly impairs the miRNA pathway. Furthermore, Nup358 interacts with AGO and GW182 proteins and promotes the association of target mRNA with miRISC A well-characterized SUMO-interacting motif (SIM) in Nup358 is sufficient for Nup358 to directly bind to AGO proteins. Moreover, AGO and PIWI proteins interact with SIMs derived from other SUMO-binding proteins. Our study indicates that Nup358-AGO interaction is important for miRNA-mediated gene silencing and identifies SIM as a new interacting motif for the AGO family of proteins. The findings also support a model wherein the coupling of miRISC with the target mRNA could occur at AL, specialized domains within the ER, and at the nuclear envelope.
Subject(s)
Argonaute Proteins/metabolism , MicroRNAs/genetics , Molecular Chaperones/metabolism , Nuclear Pore Complex Proteins/metabolism , Protein Interaction Domains and Motifs , RNA, Messenger/genetics , RNA-Induced Silencing Complex/metabolism , Amino Acid Sequence , Animals , Argonaute Proteins/chemistry , Cell Line , Gene Silencing , Humans , Intranuclear Inclusion Bodies/metabolism , MicroRNAs/metabolism , Models, Biological , Models, Molecular , Molecular Chaperones/chemistry , Nuclear Pore Complex Proteins/chemistry , Protein Binding , Protein Conformation , RNA Interference , RNA, Messenger/metabolism , Signal Transduction , Zinc FingersABSTRACT
mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3'-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2-4 d to complete (after cell lines expressing all required transgenes have been generated).
Subject(s)
Image Processing, Computer-Assisted/methods , Protein Biosynthesis/genetics , RNA, Messenger/analysis , Single Molecule Imaging/methods , HEK293 Cells , Humans , RNA, Messenger/geneticsABSTRACT
Vaccinia virus (VACV), a member of the Poxviridae family, uses cytoplasmic factories for its replication. Recent studies indicated that VACV infection requires a set of nucleoporins. However, how the nucleoporins contribute to viral life cycle remains unclear. Here, we report that the nucleoporins Nup62 and Nup358 localize to the cytoplasmic viral factories (VFs). Nup358 was targeted to the VFs at 6h post-infection (hpi), whereas Nup62, along with the previously reported translation factors such as eIF4E, eIF3η and G3BP1, was recruited to the VFs at 8 hpi. Nup358 depletion led to a decrease in the size and number of viral factories and reduction in viral yield. Further studies showed that Nup358 is involved in recruiting Nup62 and eIF4E to the VFs. Collectively, our results reveal spatio-temporal regulation in the recruitment of nucleoporins and translation factors to VFs, and particularly the importance of Nup358 in VACV infection.
Subject(s)
Gene Expression Regulation/physiology , Molecular Chaperones/metabolism , Nuclear Pore Complex Proteins/metabolism , Vaccinia virus/physiology , Virus Replication/physiology , Cell Line , DNA, Viral/genetics , DNA, Viral/metabolism , Eukaryotic Initiation Factor-4E/genetics , Eukaryotic Initiation Factor-4E/metabolism , HeLa Cells , Humans , Molecular Chaperones/genetics , Nuclear Pore Complex Proteins/genetics , Protein TransportABSTRACT
Atypical PKC (aPKC) family members are involved in regulation of diverse cellular processes, including cell polarization. aPKCs are known to be activated by phosphorylation of specific threonine residues in the activation loop and turn motif. They can also be stimulated by interaction with Cdc42~GTP-Par6 complex. Here we report that PKCζ, a member of the aPKC family, is activated by SUMOylation. We show that aPKC is endogenously modified by SUMO1 and the nucleoporin Nup358 acts as its SUMO E3 ligase. Results from in vitro SUMOylation and kinase assays showed that the modification enhances the kinase activity of PKCζ by ~10-fold. By monitoring the phosphorylation of Lethal giant larvae (Lgl), a downstream target of aPKC, we confirmed these findings in vivo. Consistent with the function of Nup358 as a SUMO E3 ligase for aPKC, depletion of Nup358 attenuated the extent of SUMOylation and the activity of aPKC. Moreover, overexpression of the C-terminal fragment of Nup358 that possesses the E3 ligase activity enhanced SUMOylation of endogenous aPKC and its kinase activity. Collectively, our studies reveal a role for Nup358-dependent SUMOylation in the regulation of aPKC activity and provide a framework for understanding the role of Nup358 in cell polarity.
ABSTRACT
Ran, a member of the Ras-GTPase superfamily, has a well-established role in regulating the transport of macromolecules across the nuclear envelope (NE). Ran has also been implicated in mitosis, cell cycle progression, and NE formation. Over-expression of Ran is associated with various cancers, although the molecular mechanism underlying this phenomenon is unclear. Serendipitously, we found that Ran possesses the ability to move from cell-to-cell when transiently expressed in mammalian cells. Moreover, we show that the inter-cellular transport of Ran is GTP-dependent. Importantly, Ran displays a similar distribution pattern in the recipient cells as that in the donor cell and co-localizes with the Ran binding protein Nup358 (also called RanBP2). Interestingly, leptomycin B, an inhibitor of CRM1-mediated export, or siRNA mediated depletion of CRM1, significantly impaired the inter-cellular transport of Ran, suggesting a function for CRM1 in this process. These novel findings indicate a possible role for Ran beyond nucleo-cytoplasmic transport, with potential implications in inter-cellular communication and cancers.