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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.

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.

mRNA localization to subcellular compartments has been reported across all kingdoms of life and it is generally believed to promote asymmetric protein synthesis and localization. In striking contrast to previous observations, we show that in S. cerevisiae the B-type cyclin CLB2 mRNA is localized and translated in the yeast bud, while the Clb2 protein, a key regulator of mitosis progression, is concentrated in the mother nucleus. Using single-molecule RNA imaging in fixed (smFISH) and living cells (MS2 system), we show that the CLB2 mRNA is transported to the yeast bud by the She2-She3 complex, via an mRNA ZIP-code situated in the coding sequence. In CLB2 mRNA localization mutants, Clb2 protein synthesis in the bud is decreased resulting in changes in cell cycle distribution and genetic instability. Altogether, we propose that CLB2 mRNA localization acts as a sensor for bud development to couple cell growth and cell cycle progression, revealing a novel function for mRNA localization.

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.

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.

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.

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.

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.

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.

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. Interestingly, 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. Here we show that Yra1 binds an HO-induced irreparable DSB. 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. Unexpectedly, while the Yra1 C-box is essential, Yra1 sumoylation and/or ubiquitination are dispensable in this process.

The first sentence under the subheading ‘Regulating developmental stages’ has been updated to clarify that the early syncytial embryos referred to are from C. elegans, and that the smFISH and MS2–MCP studies mentioned extend previous observations summarized in reference 24. These changes have been made in the HTML and PDF versions of the article.

“DNA zip codes” control positioning and interchromosomal clustering of GAL1-10 in yeast. However, these two phenomena have distinct molecular mechanisms, requiring different nuclear pore proteins, and are regulated differently by transcription and the cell cycle., 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.

Mitochondria are dynamic in their size and morphology yet must also precisely control their protein composition according to cellular energy demand. This control is particularly complicated for mitochondria, as they must coordinate gene expression from both the nuclear and mitochondrial genome. We have found that cells are able to use this dynamic morphology to post-transcriptionally coordinate protein expression with the metabolic demands of the cell through enhanced mRNA localization to the mitochondria. As yeast switch to respiratory metabolism, they increase their mitochondrial volume fraction that is, the ratio of mitochondrial volume to intracellular volume which drives the localization of nuclear-encoded mitochondrial mRNAs to the surface of the mitochondria. Through artificial tethering experiments, we show that this mitochondrial localization is sufficient to increase protein production, whereas sequestering mRNAs away from the mitochondrial surface decreases protein production, and those cells are deficient in growth in respiratory conditions. Furthermore, we find that this mRNA sensitivity to mitochondrial volume fraction is driven by the speed of translation downstream of the mitochondrial targeting sequence (MTS), as local ribosome stalling through a stretch of polyprolines in the nascent peptide can drive constitutive localization of mRNAs to the mitochondria. This points to a mechanism by which organelle volume fraction provides feedback to regulate organelle-specific gene expression through mRNA localization while potentially circumventing the need to directly coordinate with the nuclear genome.

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 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.

Platelet-derived growth factor B (PDGF-B) overexpression induces gliomas of different grades from murine embryonic neural progenitors. For the first time, we formally demonstrated that PDGF-B-induced neoplasms undergo progression from nontumorigenic low-grade tumors toward highly malignant forms. This result, showing that PDGF-B signaling alone is insufficient to confer malignancy to cells, entails the requirement for further molecular lesions in this process. Our results indicate that one of these lesions is represented by the down-regulation of the oncosuppressor Btg2. By in vivo transplantation assays, we further demonstrate that fully progressed tumors are PDGF-B-addicted because their tumor-propagating ability is lost when the PDGF-B transgene is silenced, whereas it is promptly reacquired after its reactivation. We provide evidence that this oncogene addiction is not caused by the need for PDGF-B as a mitogen but, rather, to the fact that PDGF-B is required to overcome cell-cell contact inhibition and to confer in vivo infiltrating potential on tumor cells.

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.

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.

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.