The human genome has many short tandem repeats, yet the normal functions of these repeats are unclear. The 5\' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene contains polymorphic CGG repeats, the length of which has differing effects on FMR1 expression and human health, including the neurodevelopmental disorder fragile X syndrome. We deleted the CGG repeats in the FMR1 gene (0CGG) in human stem cells and examined the effects on differentiated neurons. 0CGG neurons have altered subcellular localization of FMR1 mRNA and protein, and differential expression of cellular stress proteins compared with neurons with normal repeats (31CGG). In addition, 0CGG neurons have altered responses to glucocorticoid receptor (GR) activation, including FMR1 mRNA localization, GR chaperone HSP90α expression, GR localization, and cellular stress protein levels. Therefore, the CGG repeats in the FMR1 gene are important for the homeostatic responses of neurons to stress signals.

The frequent exchange of mobile genetic elements (MGEs) between bacteria accelerates the spread of functional traits, including antimicrobial resistance, within the human microbiome. Yet, progress in understanding these intricate processes has been hindered by the lack of tools to map the spatial spread of MGEs in complex microbial communities, and to associate MGEs to their bacterial hosts. To overcome this challenge, we present an imaging approach that pairs single molecule DNA Fluorescence In Situ Hybridization (FISH) with multiplexed ribosomal RNA FISH, thereby enabling the simultaneous visualization of both MGEs and host bacterial taxa. We used this methodology to spatially map bacteriophage and antimicrobial resistance (AMR) plasmids in human oral biofilms, and we studied the heterogeneity in their spatial distributions and demonstrated the ability to identify their host taxa. Our data revealed distinct clusters of both AMR plasmids and prophage, coinciding with densely packed regions of host bacteria in the biofilm. These results suggest the existence of specialized niches that maintain MGEs within the community, possibly acting as local hotspots for horizontal gene transfer. The methods introduced here can help advance the study of MGE ecology and address pressing questions regarding antimicrobial resistance and phage therapy.

The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by important known stem cell-extrinsic signals. However, the cell-intrinsic mechanisms that control the distinctive proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, and heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates.
The brain is a highly complex organ made up of huge numbers of different cell types that connect up to form a precise network. All these different cell types are generated from the repeated division of a relatively small pool of cells called neural stem cells. The division of these cells needs to be carefully regulated so that the correct number and type of nerve cells are produced at the right time and place. But it remains unclear how the division rate of individual neural stem cells is controlled during development. Controlling these divisions requires the activity of countless genes to be tightly regulated over space and time. When a gene is active, it is copied via a process called transcription into a single-stranded molecule known as messenger RNA (or mRNA for short). This molecule provides the instructions needed to build the protein encoded within the gene. Proteins are the functional building blocks of all cells. The conventional way of controlling protein levels is to vary the number of mRNA molecules made by transcription. Now, Samuels et al. reveal a second mechanism of determining protein levels in the brain, through regulating the stability of mRNA after it is transcribed. Samuels et al. discovered that a key regulatory protein called Imp controls the growth and division of individual neural stem cells in the brains of developing fruit flies. The experiments showed that Imp binds to mRNA molecules that contain the code for a protein called Myc, which is known to drive cell growth and division in many different cell types. Both human Imp and Myc have been implicated in cancer. Using a technique that images single molecules of mRNA, Samuels et al. showed that the Imp protein in stem cells stabilises the mRNA molecule coding for Myc. This means that when more Imp is present, more Myc protein gets produced. Thus, the level of Imp in each individual neural stem cell fine-tunes the rate at which the cell grows and divides: the higher the level of Imp, the larger the stem cell and the faster it divides. These findings underscore how important post-transcriptional processes are for regulating gene activity in the developing brain. The methods used in this study to study mRNA molecules in single cells also provide new insights that could not be derived from the average measurements of many cells. Similar methods could also be applied to other developmental systems in the future.

In essentially every cell, proteins are asymmetrically distributed according to their function. For many genes, this protein sorting problem is solved by transporting RNA molecules encoding the protein, rather than the protein itself, to the desired subcellular location. The protein is then translated on-site to immediately produce a correctly localized protein. This strategy is widely used as thousands of RNAs localize to distinct locations across diverse cell types and species. One of the fundamental challenges to study this process is the determination of the subcellular spatial distribution of any given RNA. The number of tools available for the study of RNA localization, from classical and state-of-the-art methods for the visualization of individual RNA molecules within cells to the profiling of localized transcriptomes, is rapidly growing. These include imaging-based approaches, a variety of biochemical and mechanical fractionation techniques, and proximity-labeling methods. These procedures allow for both the detailed study of the molecular requirements for the localization of individual RNA molecules and computational studies of RNA transport on a genomic scale. Together, they have the ability to allow insight into the regulatory principles that govern the localization of diverse RNAs. These new techniques provide the framework for integrating our knowledge of the regulation of RNA localization with that of other posttranscriptional processes. This article is categorized under: RNA Export and Localization > RNA Localization RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Methods > RNA Analyses in Cells.

Engineered nanoparticles (NPs) can negatively impact biological systems through induced generation of reactive oxygen species (ROS). Overproduced ROS cause biochemical damage and hence need to be effectively buffered by a sophisticated cellular oxidative stress response system. How this complex cellular system, which consists of multiple enzymes, responds to NP-induced ROS is largely unknown. Here, we apply a single cell analysis to quantitatively evaluate 10 key ROS responsive genes simultaneously to understand how the cell prioritizes tasks and reallocates resources in response to NP-induced oxidative stress. We focus on rainbow trout gill epithelial cells-a model cell type for environmental exposure-and their response to the massive generation of ROS induced by lithium cobalt oxide (LCO) NPs, which are extensively used as cathode materials in lithium ion batteries. Using multiplexed fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) in single cells, we found a shift in the expression of oxidative stress response genes with initial increase in genes targeting superoxide species, followed by increase in genes targeting peroxide and hydroxyl species. In contrast, Li+ and Co2+, at concentrations expected to be shed from the NPs, did not induce ROS generation but showed a potent inhibition of transcription for all 10 stress response genes. Taken together, our findings suggest a \"two-hit\" model for LCO NP toxicity, where the intact LCO NPs induce high levels of ROS that elicit sequential engagement of stress response genes, while the released metal ions suppress the expression of these genes. Consequently, these effects synergistically drive the exposed cells to become more vulnerable to ROS stress and damage.

Stress granules are dynamic, conserved non-translating RNA-protein assemblies that form during cellular stress and are related to pathological aggregates in many neurodegenerative diseases. Mammalian stress granules contain stable structures, referred to as \"cores\" that can be biochemically purified. Herein, we describe a step-by-step guide on how to isolate RNA from stress granule cores for RNA-Seq analysis. We also describe a methodology for validating the RNA-Seq results by single molecule FISH and how to quantify the single molecule FISH results. These protocols provide a starting point for describing the RNA content of stress granules and may assist in the discovery of the assembly mechanisms and functions of stress granules in a variety of biological contexts.

Stress granules are mRNA-protein assemblies formed from nontranslating mRNAs. Stress granules are important in the stress response and may contribute to some degenerative diseases. Here, we describe the stress granule transcriptome of yeast and mammalian cells through RNA-sequencing (RNA-seq) analysis of purified stress granule cores and single-molecule fluorescence in situ hybridization (smFISH) validation. While essentially every mRNA, and some noncoding RNAs (ncRNAs), can be targeted to stress granules, the targeting efficiency varies from <1% to >95%. mRNA accumulation in stress granules correlates with longer coding and UTR regions and poor translatability. Quantifying the RNA-seq analysis by smFISH reveals that only 10% of bulk mRNA molecules accumulate in mammalian stress granules and that only 185 genes have more than 50% of their mRNA molecules in stress granules. These results suggest that stress granules may not represent a specific biological program of messenger ribonucleoprotein (mRNP) assembly, but instead form by condensation of nontranslating mRNPs in proportion to their length and lack of association with ribosomes.

Long noncoding RNAs (lncRNAs) have been implicated in many biological processes. However, due to the unique nature of lncRNAs and the consequential difficulties associated with their characterization, there is a growing disparity between the rate at which lncRNAs are being discovered and the assignment of biological function to these transcripts. Here we present a molecular biology toolbox equipped to help dissect aspects of lncRNA biology and reveal functionality. We outline an approach that begins with a broad survey of genome-wide, high-throughput datasets to identify potential lncRNA candidates and then narrow the focus on specific methods that are well suited to interrogate the transcripts of interest more closely. This involves the use of imaging-based strategies to validate these candidates and observe the behaviors of these transcripts at single molecule resolution in individual cells. We also describe the use of gene editing tools and interactome capture techniques to interrogate functionality and infer mechanism, respectively. With the emergence of lncRNAs as important molecules in healthy and diseased cellular function, it remains crucial to deepen our understanding of their biology.

Transcriptional regulation of gene expression is fundamental to most cellular processes, including determination of cellular fates. Quantitative studies of transcription in cultured cells have led to significant advances in identifying mechanisms underlying transcriptional control. Recent progress allowed implementation of these same quantitative methods in multicellular organisms to ask how transcriptional regulation unfolds both in vivo and at the single molecule level in the context of embryonic development. Here we review some of these advances in early Drosophila development, which bring the embryo on par with its single celled counterparts. In particular, we discuss progress in methods to measure mRNA and protein distributions in fixed and living embryos, and we highlight some initial applications that lead to fundamental new insights about molecular transcription processes. We end with an outlook on how to further exploit the unique advantages that come with investigating transcriptional control in the multicellular context of development.