Intracellular mRNA transport and localized translation.

Fine-tuning cellular physiology in response to intracellular and environmental cues requires precise temporal and spatial control of gene expression. High-resolution imaging technologies to detect mRNAs and their translation state have revealed that all living organisms localize mRNAs in subcellular compartments and create translation hotspots, enabling cells to tune gene expression locally. Therefore, mRNA localization is a conserved and integral part of gene expression regulation from prokaryotic to eukaryotic cells. In this Review, we discuss the mechanisms of mRNA transport and local mRNA translation across the kingdoms of life and at organellar, subcellular and multicellular resolution. We also discuss the properties of messenger ribonucleoprotein and higher order RNA granules and how they may influence mRNA transport and local protein synthesis. Finally, we summarize the technological developments that allow us to study mRNA localization and local translation through the simultaneous detection of mRNAs and proteins in single cells, mRNA and nascent protein single-molecule imaging, and bulk RNA and protein detection methods.

Keeping mRNPs in check during assembly and nuclear export.

The cell nucleus is an intricate organelle that coordinates multiple activities that are associated with DNA replication and gene expression. In all eukaryotes, it stores the genetic information and the machineries that control the production of mature and export-competent messenger ribonucleoproteins (mRNPs), a multistep process that is regulated in a spatial and temporal manner. Recent studies suggest that post-translational modifications play a part in coordinating the co-transcriptional assembly, remodelling and export of mRNP complexes through nuclear pores, adding a new level of regulation to the process of gene expression.

Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.

Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.

An improved MS2 system for accurate reporting of the mRNA life cycle.

The MS2-MCP system enables researchers to image multiple steps of the mRNA life cycle with high temporal and spatial resolution. However, for short-lived mRNAs, the tight binding of the MS2 coat protein (MCP) to the MS2 binding sites (MBS) protects the RNA from being efficiently degraded, and this confounds the study of mRNA regulation. Here, we describe a reporter system (MBSV6) with reduced affinity for the MCP, which allows mRNA degradation while preserving single-molecule detection determined by single-molecule FISH (smFISH) or live imaging. Constitutive mRNAs (MDN1 and DOA1) and highly-regulated mRNAs (GAL1 and ASH1) endogenously tagged with MBSV6 in Saccharomyces cerevisiae degrade normally. As a result, short-lived mRNAs were imaged throughout their complete life cycle. The MBSV6 reporter revealed that, in contrast to previous findings, coordinated recruitment of mRNAs at specialized structures such as P-bodies during stress did not occur, and mRNA degradation was heterogeneously distributed in the cytoplasm.

Ubiquitin-mediated mRNP dynamics and surveillance prior to budding yeast mRNA export.

The evolutionarily conserved mRNA export receptor Mex67/NXF1 associates with mRNAs through its adaptor, Yra1/REF, allowing mRNA ribonucleoprotein (mRNP) exit through nuclear pores. However, alternate adaptors should exist, since Yra1 is dispensable for mRNA export in Drosophila and Caenorhabditis elegans. Here we report that Mex67 interacts directly with Nab2, an essential shuttling mRNA-binding protein required for export. We further show that Yra1 enhances the interaction between Nab2 and Mex67, and becomes dispensable in cells overexpressing Nab2 or Mex67. These observations appoint Nab2 as a potential adaptor for Mex67, and define Yra1/REF as a cofactor stabilizing the adaptor-receptor interaction. Importantly, Yra1 ubiquitination by the E3 ligase Tom1 promotes its dissociation from mRNP before export. Finally, loss of perinuclear Mlp proteins suppresses the growth defects of Tom1 and Yra1 ubiquitination mutants, suggesting that Tom1-mediated dissociation of Yra1 from Nab2-bound mRNAs is part of a surveillance mechanism at the pore, ensuring export of mature mRNPs only.

Single-Molecule Fluorescent In Situ Hybridization (smFISH) for RNA Detection in the Fungal Pathogen Candida albicans.

Candida albicans is the most prevalent human fungal pathogen. Its pathogenicity is linked to the ability of C. albicans to reversibly change morphology and to grow as yeast, pseudohyphae, or hyphal cells in response to environmental stimuli. Understanding the molecular regulation controlling those morphological switches remains a challenge that, if solved, could help eradicate C. albicans infections.While numerous studies investigated gene expression changes occurring during C. albicans morphological switches using bulk approaches (e.g., RNA sequencing), here we describe a single-cell and single-molecule RNA imaging and analysis protocol to measure absolute mRNA counts in morphologically intact cells. To detect endogenous mRNAs in single fixed cells, we optimized a single-molecule fluorescent in situ hybridization (smFISH) protocol for C. albicans, which allows one to quantify the differential expression of mRNAs in yeast, pseudohyphae, or hyphal cells. We quantified the expression of two mRNAs, a cell cycle-controlled mRNA (CLB2) and a transcription factor (EFG1), which show expression changes in the different morphological cell types and nutrient conditions. In this protocol, we described in detail the major steps of this approach: growth and fixation, hybridization, imaging, cell segmentation, and mRNA spot analysis. Raw data is provided with the protocol to favor reproducibility. This approach could benefit the molecular characterization of C. albicans and other filamentous fungi, pathogenic or nonpathogenic.

cAMP in budding yeast: Also a messenger for sucrose metabolism?

S. cerevisiae (or budding yeast) is an important micro-organism for sucrose-based fermentation in biotechnology. Yet, it is largely unknown how budding yeast adapts to sucrose transitions. Sucrose can only be metabolized when the invertase or the maltose machinery are expressed and we propose that the Gpr1p receptor signals extracellular sucrose availability via the cAMP peak to adapt cells accordingly. A transition to sucrose or glucose gave a transient cAMP peak which was maximally induced for sucrose. When transitioned to sucrose, cAMP signalling mutants showed an impaired cAMP peak together with a lower growth rate, a longer lag phase and a higher final OD600 compared to a glucose transition. These effects were not caused by altered activity or expression of enzymes involved in sucrose metabolism and imply a more general metabolic adaptation defect. Basal cAMP levels were comparable among the mutant strains, suggesting that the transient cAMP peak is required to adapt cells correctly to sucrose. We propose that the short-term dynamics of the cAMP signalling cascade detects long-term extracellular sucrose availability and speculate that its function is to maintain a fermentative phenotype at continuously low glucose and fructose concentrations.

Understanding spatiotemporal coupling of gene expression using single molecule RNA imaging technologies.

Across all kingdoms of life, gene regulatory mechanisms underlie cellular adaptation to ever-changing environments. Regulation of gene expression adjusts protein synthesis and, in turn, cellular growth. Messenger RNAs are key molecules in the process of gene expression. Our ability to quantitatively measure mRNA expression in single cells has improved tremendously over the past decades. This revealed an unexpected coordination between the steps that control the life of an mRNA, from transcription to degradation. Here, we provide an overview of the state-of-the-art imaging approaches for measurement and quantitative understanding of gene expression, starting from the early visualizations of single genes by electron microscopy to current fluorescence-based approaches in single cells, including live-cell RNA-imaging approaches to FISH-based spatial transcriptomics across model organisms. We also highlight how these methods have shaped our current understanding of the spatiotemporal coupling between transcriptional and post-transcriptional events in prokaryotes. We conclude by discussing future challenges of this multidisciplinary field.Abbreviations: mRNA: messenger RNA; rRNA: ribosomal rDNA; tRNA: transfer RNA; sRNA: small RNA; FISH: fluorescence in situ hybridization; RNP: ribonucleoprotein; smFISH: single RNA molecule FISH; smiFISH: single molecule inexpensive FISH; HCR-FISH: Hybridization Chain-Reaction-FISH; RCA: Rolling Circle Amplification; seqFISH: Sequential FISH; MERFISH: Multiplexed error robust FISH; UTR: Untranslated region; RBP: RNA binding protein; FP: fluorescent protein; eGFP: enhanced GFP, MCP: MS2 coat protein; PCP: PP7 coat protein; MB: Molecular beacons; sgRNA: single guide RNA.

PDGF-B-driven gliomagenesis can occur in the absence of the proteoglycan NG2.

BACKGROUND: In the last years, the transmembrane proteoglycan NG2 has gained interest as a therapeutic target for the treatment of diverse tumor types, including gliomas, because increases of its expression correlate with dismal prognosis. NG2 has been shown to function as a co-receptor for PDGF ligands whose aberrant expression is common in gliomas. We have recently generated a glioma model based on the overexpression of PDGF-B in neural progenitors and here we investigated the possible relevance of NG2 during PDGF-driven gliomagenesis. METHODS: The survival curves of NG2-KO mice overexpressing PDGF-B were compared to controls by using a Log-rank test. The characteristics of tumors induced in NG2-KO were compared to those of tumors induced in wild type mice by immunostaining for different cell lineage markers and by transplantation assays in adult mice. RESULTS: We showed that the lack of NG2 does not appreciably affect any of the characterized steps of PDGF-driven brain tumorigenesis, such as oligodendrocyte progenitor cells (OPC) induction, the recruitment of bystander OPCs and the progression to full malignancy, which take place as in wild type animals. CONCLUSIONS: Our analysis, using both NG2-KO mice and a miRNA based silencing approach, clearly demonstrates that NG2 is not required for PDGF-B to efficiently induce and maintain gliomas from neural progenitors. On the basis of the data obtained, we therefore suggest that the role of NG2 as a target molecule for glioma treatment should be carefully reconsidered.

Simultaneous Detection of mRNA and Protein in S. cerevisiae by Single-Molecule FISH and Immunofluorescence.

Single-molecule fluorescent in situ hybridization (smFISH) enables the detection and quantification of endogenous mRNAs within intact fixed cells. This method utilizes tens of singly labeled fluorescent DNA probes hybridized against the mRNA of interest, which can be detected by using standard wide-field fluorescence microscopy. This approach provides the means to generate absolute quantifications of gene expression within single cells, which can be used to link molecular fluctuations to phenotypes. To be able to correlate the expression of an mRNA and a protein of interest in individual cells, we combined smFISH with immunofluorescence (IF) in yeast cells. Here, we present our smFISH-IF protocol to visualize and quantify two cell cycle-controlled mRNAs (CLN2 and ASH1) and the cell cycle marker alpha-tubulin in S. cerevisiae. This protocol, which is performed over 2 days, can be used to visualize up to three colors at the time (i.e., two mRNAs, one protein). Even if the described protocol is designed for S. cerevisiae, we think that the considerations discussed here can be useful to develop and troubleshoot smFISH-IF protocols for other model organisms.

Single molecule mRNA fluorescent in situ hybridization combined with immunofluorescence in S. cerevisiae: Dataset and quantification.

Single-molecule fluorescent in situ hybridization (smFISH) has emerged as a powerful technique that allows one to localize and quantify the absolute number of mRNAs in single cells. In combination with immunofluorescence (IF), smFISH can be used to correlate the expression of an mRNA and a protein of interest in single cells. Here, we provide and quantify an smFISH-IF dataset in S. cerevisiae. We measured the expression of the cell cycle-controlled mRNA CLN2 and the cell cycle marker alpha-tubulin. The smFISH-IF protocol describing the dataset generation is published in the accompanying article "Simultaneous detection of mRNA and protein in S. cerevisiae by single-molecule FISH and Immunofluorescence" [1]. Here, we analyze the smFISH data using the freely available software FISH-quant [2]. The provided datasets are intended to assist scientists interested in setting up smFISH-IF protocol in their laboratory. Furthermore, scientists interested in the generation of imaging analysis tools for single-cell approaches may find the provided dataset useful. To this end, we provide the differential interference contrast (DIC) channel, as well as multicolor, raw Z-stacks for smFISH, IF and DAPI.

New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells.

Live imaging of single RNA from birth to death brought important advances in our understanding of the spatiotemporal regulation of gene expression. These studies have provided a comprehensive understanding of RNA metabolism by describing the process step by step. Most of these studies used for live imaging a genetically encoded RNA-tagging system fused to fluorescent proteins. One of the best characterized RNA-tagging systems is derived from the bacteriophage MS2 and it allows single RNA imaging in real-time and live cells. This system has been successfully used to track the different steps of mRNA processing in many living organisms. The recent development of optimized MS2 and MCP variants now allows the labeling of endogenous RNAs and their imaging without modifying their behavior. In this chapter, we discuss the improvements in detecting single mRNAs with different variants of MCP and fluorescent proteins that we tested in yeast and mammalian cells. Moreover, we describe protocols using MS2-MCP systems improved for real-time imaging of single mRNAs and transcription dynamics in S. cerevisiae and mammalian cells, respectively.

Mitochondrial volume fraction and translation duration impact mitochondrial mRNA localization and protein synthesis.

Mitochondria are dynamic organelles that must precisely control their protein composition according to cellular energy demand. Although nuclear-encoded mRNAs can be localized to the mitochondrial surface, the importance of this localization is unclear. As yeast switch to respiratory metabolism, there is an increase in the fraction of the cytoplasm that is mitochondrial. Our data point to this change in mitochondrial volume fraction increasing the localization of certain nuclear-encoded mRNAs to the surface of the mitochondria. We show that mitochondrial mRNA localization is necessary and sufficient to increase protein production to levels required during respiratory growth. Furthermore, we find that ribosome stalling impacts mRNA sensitivity to mitochondrial volume fraction and counterintuitively leads to enhanced protein synthesis by increasing mRNA localization to mitochondria. This points to a mechanism by which cells are able to use translation elongation and the geometric constraints of the cell to fine-tune organelle-specific gene expression through mRNA localization.

PDGF-B induces a homogeneous class of oligodendrogliomas from embryonic neural progenitors.

We describe the generation of mouse gliomas following the overexpression of PDGF-B in embryonic neural progenitors. Our histopathological, immunohistochemical and genome-wide expression analyses revealed a surprising uniformity among PDGF-B induced tumors, despite they were generated by transducing a highly heterogeneous population of progenitor cells known for their ability to produce all the cell types of the central nervous system. Comparison of our microarray data with published gene expression data sets for many different murine neural cell types revealed a closest correlation between our tumor cells and oligodendrocyte progenitor cells, confirming definitively that PDGF-B-induced gliomas are pure oligodendrogliomas. Importantly, we show that this uniformity is likely due to the ability of PDGF-B overexpression to respecify competent embryonic neural precursors toward the oligodendroglial lineage, providing evidence that the transforming activity of PDGF-B is influenced by the developmental potential of the targeted cells. Interestingly, we found that PDGF-B-induced tumors harbor different proliferating cell populations. However only PDGF-B-overexpressing cells are tumorigenic, indicating that paracrine signaling from the tumor is unable to transform bystander cells.

Imaging Single mRNA Molecules in Mammalian Cells Using an Optimized MS2-MCP System.

Visualization of single mRNAs in their native cellular environment provides key information to study gene expression regulation. This fundamental biological question triggered the development of the MS2-MCP (MS2-Capsid Protein) system to tag mRNAs and image their life cycle using widefield fluorescence microscopy. The last two decades have evolved toward improving the qualitative and quantitative characteristics of the MS2-MCP system. Here, we provide a protocol to use the latest versions, MS2V6 and MS2V7, to tag and visualize mRNAs in mammalian cells in culture. The motivation behind engineering MS2V6 and MS2V7 was to overcome a degradation caveat observed in S. cerevisiae with the previous MS2-MCP systems. While for yeast we recommend the use of MS2V6, we found that for live-cell imaging experiments in mammalian cells, the MS2V7 has improved reporter properties.

The mRNA export adaptor Yra1 contributes to DNA double-strand break repair through its C-box domain.

Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S. cerevisiae. Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism. Yra1 was also identified at an HO-induced DSB and Yra1 depletion causes defects in DSB repair. Previous work from our laboratory showed that Yra1 ubiquitination by Tom1 is important for mRNA export. Here, we found that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-Slx8 implicated in the interaction of irreparable DSB with nuclear pores. We further show that Yra1 binds an HO-induced irreparable DSB in a process dependent on resection. Importantly, a Yra1 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-induced irreparable DSB and becomes lethal under DSB induction in a HO-cut reparable system. Together, the data provide evidence that Yra1 plays a crucial role in DSB repair via homologous recombination. While Yra1 sumoylation and/or ubiquitination are dispensable, the Yra1 C-box region is essential in this process.

Single-mRNA detection in living S. cerevisiae using a re-engineered MS2 system.

The MS2 system has been widely used, in organisms ranging from bacteria to higher eukaryotes, to image single mRNAs in intact cells with high precision. We have recently re-engineered the MS2 system for accurate detection of mRNAs in living Saccharomyces cerevisiae. Previous MS2 systems affected the degradation of the tagged mRNA, which led to accumulation of MS2 fragments and to erroneous conclusions about mRNA localization and expression. Here we describe a step-by-step protocol for the use of our latest MS2 system (MBSV6) for detecting endogenously tagged mRNAs using wide-field fluorescent microscopy in living yeast. The procedure is divided into three stages: tagging of endogenous gene with MBSV6 (~2 weeks), a two-color single-molecule RNA fluorescent in situ hybridization (smFISH) procedure to quantitatively assess whether mRNAs tagged with MS2 and MS2-coat protein (MCP) behave like untagged mRNAs (2 d, plus additional time for quantification), and a procedure to quantify single mRNAs by live imaging using wide-field microscopy (1 d, plus additional time for quantification). With this method it is now possible to interrogate all phases of mRNA expression, from transcription through decay. The described protocol is designed for S. cerevisiae; however, we think that our approach and the considerations discussed here can be extended to Escherichia coli, Drosophila, Caenorhabditis elegans, and mammalian cells.

Imaging mRNA In Vivo, from Birth to Death.

RNA is the fundamental information transfer system in the cell. The ability to follow single messenger RNAs (mRNAs) from transcription to degradation with fluorescent probes gives quantitative information about how the information is transferred from DNA to proteins. This review focuses on the latest technological developments in the field of single-mRNA detection and their usage to study gene expression in both fixed and live cells. By describing the application of these imaging tools, we follow the journey of mRNA from transcription to decay in single cells, with single-molecule resolution. We review current theoretical models for describing transcription and translation that were generated by single-molecule and single-cell studies. These methods provide a basis to study how single-molecule interactions generate phenotypes, fundamentally changing our understating of gene expression regulation.

Subnuclear positioning and interchromosomal clustering of the GAL1-10 locus are controlled by separable, interdependent mechanisms.

On activation, the GAL genes in yeast are targeted to the nuclear periphery through interaction with the nuclear pore complex. Here we identify two cis-acting "DNA zip codes" from the GAL1-10 promoter that are necessary and sufficient to induce repositioning to the nuclear periphery. One of these zip codes, GRS4, is also necessary and sufficient to promote clustering of GAL1-10 alleles. GRS4, and to a lesser extent GRS5, contribute to stronger expression of GAL1 and GAL10 by increasing the fraction of cells that respond to the inducer. The molecular mechanism controlling targeting to the NPC is distinct from the molecular mechanism controlling interchromosomal clustering. Targeting to the nuclear periphery and interaction with the nuclear pore complex are prerequisites for gene clustering. However, once formed, clustering can be maintained in the nucleoplasm, requires distinct nuclear pore proteins, and is regulated differently through the cell cycle. In addition, whereas targeting of genes to the NPC is independent of transcription, interchromosomal clustering requires transcription. These results argue that zip code-dependent gene positioning at the nuclear periphery and interchromosomal clustering represent interdependent phenomena with distinct molecular mechanisms.