Single-Molecule Imaging Uncovers Rules Governing Nonsense-Mediated mRNA Decay

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Inter-cellular transport of ran GTPase.

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

Inter-cellular transfer of Ran.

<p>HeLa cells were transfected with indicated constructs for 9 h and were then co-cultured with untransfected NIH3T3 cells for 18 h. Cells were stained with GFP antibody (green) and the DNA dye Hoechst 33342 (pseudocoloured in red). Arrows indicate NIH3T3 cells as detected by the characteristic punctate staining of the nucleus. Scale bar, 25 μm.</p

Ectopic expression of Ran GTPase.

<p>HeLa cells were transfected with indicated constructs for 24 h and fixed using methanol (A) or paraformaldehyde (B) and were processed for fluorescence microscopy. GFP is detected with a specific polyclonal antibody (green) and DNA was stained with Hoechst 33342 (blue). (C) COS-7 cells were transfected with indicated constructs for 24 h and the unfixed cells were directly visualized under fluorescence microscope. In all the above experiments, the adjacent respective graph represents quantitative data indicating the percentage of cells showing the GFP proteins and was derived from three independent experiments (in each experiment at least 100 cells were counted). Data are expressed as mean ± SD. Scale bar, 20 μm. (D) HeLa cells transfected with indicated constructs were lysed, separated on 10% SDS-PAGE and analysed by western blotting (WB) with GFP and Ran antibodies. α-tubulin was used as loading control. Molecular weights (in kDa) are shown in numbers.</p

Live imaging of mRNA using RNA-stabilized fluorogenic proteins

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

Quantification of mRNA translation in live cells using single-molecule imaging

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, 3ʹ-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)

Transient transfection assay for inter-cellular transport of Ran.

<p>HeLa cells were co-transfected with mCherry-α-tubulin (transfection marker, red) and indicated GFP constructs (green) for 9 h. Cells were fixed and analysed for the presence of mCherry and GFP. DNA was stained in blue. Scale bar, 20 μm. Quantitative data showing the number of recipient cells displaying GFP staining surrounding the mCherry-α-tubulin positive donor cell. Cells were counted from 15 individual fields randomly across three independent experiments. Data are expressed as mean ± SD.</p

Quantification of mRNA translation in live cells using single-molecule imaging

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, 3ʹ-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)

Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding

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. The MoonTag system is a fluorescence labeling system for visualizing translation of single mRNA molecules in live cells. Combining the MoonTag system with the orthogonal SunTag system enables simultaneous measurements of translation of two open reading frames in an mRNA and reveals that ribosomes differentially decode individual mRNA molecules

HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion

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.An Open Targets grant [OTAR2054], the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council (MRC), Alzheimer’s Society and Alzheimer’s Research UK