ABSTRACT
Women and racial minorities are underrepresented in the synthetic biology community. Developing a scholarly identity by engaging in a scientific community through writing and communication is an important component for STEM retention, particularly for underrepresented individuals. Several excellent pedagogical tools have been developed to teach scientific literacy and to measure competency in reading and interpreting scientific literature. However, fewer tools exist to measure learning gains with respect to writing, or that teach the more abstract processes of peer review and scientific publishing, which are essential for developing scholarly identity and publication currency. Here we describe our approach to teaching scientific writing and publishing to undergraduate students within a synthetic biology course. Using gold standard practices in project-based learning, we created a writing project in which students became experts in a specific application area of synthetic biology with relevance to an important global problem or challenge. To measure learning gains associated with our learning outcomes, we adapted and expanded the Student Attitudes, Abilities, and Beliefs (SAAB) concept inventory to include additional questions about the process of scientific writing, authorship, and peer review. Our results suggest the project-based approach was effective in achieving the learning objectives with respect to writing and peer reviewed publication, and resulted in high student satisfaction and student self-reported learning gains. We propose that these educational practices could contribute directly to the development of scientific identity of undergraduate students as synthetic biologists, and will be useful in creating a more diverse synthetic biology research enterprise.
ABSTRACT
Lead (Pb(II)) is a pervasive heavy metal toxin with many well-established negative effects on human health. Lead toxicity arises from cumulative, repeated environmental exposures. Thus, prophylactic strategies to protect against the bioaccumulation of lead could reduce lead-associated human pathologies. Here we show that DNA and RNA aptamers protect C. elegans from toxic phenotypes caused by lead. Reproductive toxicity, as measured by brood size assays, is prevented by co-feeding of animals with DNA or RNA aptamers. Similarly, lead-induced neurotoxicity, measured by behavioral assays, are also normalized by aptamer feeding. Further, cultured human HEK293 and primary murine osteoblasts are protected from lead toxicity by transfection with DNA aptamers. The osteogenic development, which is decreased by lead exposure, is maintained by prior transfection of lead-binding DNA aptamers. Aptamers may be an effective strategy for the protection of human health in the face of increasing environmental toxicants.
ABSTRACT
Lead (Pb(II)) is a pervasive heavy metal toxin with many well-established negative effects on human health. Lead toxicity arises from cumulative, repeated environmental exposures. Thus, prophylactic strategies to protect against the bioaccumulation of lead could reduce lead-associated human pathologies. Here we show that DNA and RNA aptamers protect C. elegans from toxic phenotypes caused by lead. Reproductive toxicity, as measured by brood size assays, is prevented by co-feeding of animals with DNA or RNA aptamers. Similarly, lead-induced behavioral anomalies are also normalized by aptamer feeding. Further, cultured human HEK293 and primary murine osteoblasts are protected from lead toxicity by transfection with DNA aptamers. The osteogenic development, which is decreased by lead exposure, is maintained by prior transfection of lead-binding DNA aptamers. Aptamers may be an effective strategy for the protection of human health in the face of increasing environmental toxicants.
ABSTRACT
Cellular senescence increases with aging. While senescence is associated with an exit of the cell cycle, there is ample evidence that post-mitotic cells including neurons can undergo senescence as the brain ages, and that senescence likely contributes significantly to the progression of neurodegenerative diseases (ND) such as Alzheimer's Disease (AD) and Amyotrophic Lateral Sclerosis (ALS). Stress granules (SGs) are stress-induced cytoplasmic biomolecular condensates of RNA and proteins, which have been linked to the development of AD and ALS. The SG seeding hypothesis of NDs proposes that chronic stress in aging neurons results in static SGs that progress into pathological aggregates Alterations in SG dynamics have also been linked to senescence, though studies that link SGs and senescence in the context of NDs and the aging brain have not yet been performed. In this Review, we summarize the literature on senescence, and explore the contribution of senescence to the aging brain. We describe senescence phenotypes in aging neurons and glia, and their links to neuroinflammation and the development of AD and ALS. We further examine the relationships of SGs to senescence and to ND. We propose a new hypothesis that neuronal senescence may contribute to the mechanism of SG seeding in ND by altering SG dynamics in aged cells, thereby providing additional aggregation opportunities within aged neurons.
Subject(s)
Amyotrophic Lateral Sclerosis , Neurodegenerative Diseases , Humans , Amyotrophic Lateral Sclerosis/genetics , Stress Granules , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/pathology , Neurons/metabolism , Proteins/metabolism , Neurodegenerative Diseases/metabolismABSTRACT
Inducible bacterial promoters are ubiquitous biotechnology tools that have a consistent architecture including two key elements: the operator region recognized by the transcriptional regulatory proteins, and the -10 and -35 consensus sequences required to recruit the sigma (σ) 70 subunits of RNA polymerase to initiate transcription. Despite their widespread use, leaky transcription in the OFF state remains a challenge. We have updated the architecture of the lac and tet promoters to improve their strength, control and portability by the adaptation of the consensus -10 and -35 sequence boxes strongly targeted by σ70 , incorporation of a strong ribosome binding site recognized broadly by Gram-negative bacteria, and independent control of the transcriptional regulators by constitutive promoters. To test the promoters, we use the far-red fluorescent protein mCardinal, which significantly improves the signal-to-background ratio of promoter measurements over widely utilized green fluorescent proteins. We validate the improvement in OFF state control and inducibility by demonstrating production of the toxic and aggregate-prone cocaine esterase enzyme CocE. We further demonstrate portability of the promoters to additional Gram-negative species Pseudomonas putida and Vibrio natriegens. Our results represent a significant improvement over existing protein expression systems that will enable advances in protein production for various biotechnology applications.
Subject(s)
DNA-Directed RNA Polymerases , Transcription Factors , Promoter Regions, Genetic , Transcription Factors/metabolism , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/metabolism , Bacteria/metabolism , Gram-Negative Bacteria/genetics , Gram-Negative Bacteria/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Transcription, Genetic , Sigma Factor/genetics , Sigma Factor/metabolism , Gene Expression Regulation, BacterialABSTRACT
Stress granules (SGs) are non-membrane bound cytoplasmic condensates that form in response to a variety of different stressors. Canonical SGs are thought to have a cytoprotective role, reallocating cellular resources during stress by activation of the integrated stress response (ISR) to inhibit translation and avoid apoptosis. However, different stresses result in compositionally distinct, non-canonical SG formation that is likely pro-apoptotic, though the exact function(s) of both SGs subtypes remain unclear. A unique non-canonical SG subtype is triggered upon exposure to ultraviolet (UV) radiation. While it is generally agreed that UV SGs are bona fide SGs due to their dependence upon the core SG nucleating protein Ras GTPase-activating protein-binding protein 1 (G3BP1), the localization of other key components of UV SGs are unknown or under debate. Further, the dynamics of UV SGs are not known, though unique properties such as cell cycle dependence have been observed. This Perspective compiles the available information on SG subtypes and on UV SGs in particular in an attempt to understand the formation, dynamics, and function of these mysterious stress-specific complexes. We identify key gaps in knowledge related to UV SGs, and examine the unique aspects of their formation. We propose that more thorough knowledge of the distinct properties of UV SGs will lead to new avenues of understanding of the function of SGs, as well as their roles in disease.
ABSTRACT
Reproducibility is a key challenge of synthetic biology, but the foundation of reproducibility is only as solid as the reference materials it is built upon. Here we focus on the reproducibility of fluorescence measurements from bacteria transformed with engineered genetic constructs. This comparative analysis comprises three large interlaboratory studies using flow cytometry and plate readers, identical genetic constructs, and compatible unit calibration protocols. Across all three studies, we find similarly high precision in the calibrants used for plate readers. We also find that fluorescence measurements agree closely across the flow cytometry results and two years of plate reader results, with an average standard deviation of 1.52-fold, while the third year of plate reader results are consistently shifted by more than an order of magnitude, with an average shift of 28.9-fold. Analyzing possible sources of error indicates this shift is due to incorrect preparation of the fluorescein calibrant. These findings suggest that measuring fluorescence from engineered constructs is highly reproducible, but also that there is a critical need for access to quality controlled fluorescent calibrants for plate readers.
Subject(s)
Bacteria/genetics , Genetic Engineering/methods , Calibration , Flow Cytometry/methods , Fluorescence , Reproducibility of Results , Synthetic Biology/methodsABSTRACT
Bisphenol-A (BPA) is a ubiquitous precursor of polycarbonate plastics that is found in the blood and serum of >92% of Americans. While BPA has been well documented to act as a weak estrogen receptor (ER) agonist, its effects on cellular stress are unclear. Here, we demonstrate that high-dose BPA causes stress granules (SGs) in human cells. A common estrogen derivative, ß-estradiol, does not trigger SGs, indicating the mechanism of SG induction is not via the ER pathway. We also tested other structurally related environmental contaminants including the common BPA substitutes BPS and BPF, the industrial chemical 4-nonylphenol (4-NP) and structurally related compounds 4-EP and 4-VP, as well as the pesticide 2,4-dichlorophenoxyacetic acid (2,4-D). The variable results from these related compounds suggest that structural homology is not a reliable predictor of the capacity of a compound to cause SGs. Also, we demonstrate that BPA acts primarily through the PERK pathway to generate canonical SGs. Finally, we show that chronic exposure to a low physiologically relevant dose of BPA suppresses SG assembly upon subsequent acute stress. Interestingly, this SG inhibition does not affect phosphorylation of eIF2α or translation inhibition, thus uncoupling the physical assembly of SGs from translational control. Our work identifies additional effects of BPA beyond endocrine disruption that may have consequences for human health.
Subject(s)
Benzhydryl Compounds/metabolism , Phenols/metabolism , Stress Granules/metabolism , Stress, Physiological , Animals , Benzhydryl Compounds/pharmacology , Cell Line , Eukaryotic Initiation Factor-2/metabolism , Gene Expression Regulation , Mice , Phenols/pharmacology , eIF-2 Kinase/metabolismABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
Subject(s)
Bacterial Load/genetics , Escherichia coli/growth & development , Flow Cytometry , Calibration , Cell Count/methods , Escherichia coli/genetics , Fluorescence , Gene Expression Regulation, Bacterial/geneticsABSTRACT
Synthetic biology is a rapidly growing field defined more by its process and values than by experimental goals. These values include the importance of diverse teams, open science, and the power of novice perspectives. Educating the next generation of synthetic biologists means teaching its values as well as its processes.
Subject(s)
Synthetic Biology/education , Teaching/trends , HumansABSTRACT
For synthetic biology to mature, composition of devices into functional systems must become routine. This requires widespread adoption of comparable and replicable units of measurement. Interlaboratory studies organized through the International Genetically Engineered Machine (iGEM) competition show that fluorescence can be calibrated with simple, low-cost protocols, so fluorescence should no longer be published without units.
Subject(s)
Genetic Engineering/standards , Laboratory Proficiency Testing/organization & administration , Spectrometry, Fluorescence/standards , Synthetic Biology/standards , Base Sequence , DNA/analysis , DNA/genetics , DNA/metabolism , Genetic Engineering/instrumentation , Genetic Engineering/methods , Humans , Synthetic Biology/instrumentation , Synthetic Biology/methodsABSTRACT
The RNA binding proteins Rbfox1/2/3 regulate alternative splicing in the nervous system, and disruption of Rbfox1 has been implicated in autism. However, comprehensive identification of functional Rbfox targets has been challenging. Here, we perform HITS-CLIP for all three Rbfox family members in order to globally map, at a single-nucleotide resolution, their in vivo RNA interaction sites in the mouse brain. We find that the two guanines in the Rbfox binding motif UGCAUG are critical for protein-RNA interactions and crosslinking. Using integrative modeling, these interaction sites, combined with additional datasets, define 1,059 direct Rbfox target alternative splicing events. Over half of the quantifiable targets show dynamic changes during brain development. Of particular interest are 111 events from 48 candidate autism-susceptibility genes, including syndromic autism genes Shank3, Cacna1c, and Tsc2. Alteration of Rbfox targets in some autistic brains is correlated with downregulation of all three Rbfox proteins, supporting the potential clinical relevance of the splicing-regulatory network.
Subject(s)
Autistic Disorder/genetics , Brain/growth & development , Gene Regulatory Networks , RNA-Binding Proteins/genetics , RNA/genetics , Repressor Proteins/genetics , Alternative Splicing , Animals , Autistic Disorder/metabolism , Base Sequence , Brain/metabolism , Exons , Genetic Predisposition to Disease , Humans , Immunoprecipitation , Mice , Models, Genetic , Models, Molecular , Molecular Sequence Data , RNA/metabolism , RNA-Binding Proteins/metabolism , Repressor Proteins/metabolismABSTRACT
Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.
Subject(s)
Fragile X Mental Retardation Protein/genetics , Fragile X Mental Retardation Protein/physiology , Fragile X Syndrome/genetics , Fragile X Syndrome/physiopathology , Transcription Factors/deficiency , Transcription Factors/genetics , Transcription Factors/physiology , mRNA Cleavage and Polyadenylation Factors/deficiency , mRNA Cleavage and Polyadenylation Factors/genetics , mRNA Cleavage and Polyadenylation Factors/physiology , 3' Untranslated Regions , Animals , Disease Models, Animal , Fragile X Syndrome/psychology , Hippocampus/physiopathology , Humans , Male , Memory, Short-Term/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Multidimensional cancer genome analysis and validation has defined Quaking (QKI), a member of the signal transduction and activation of RNA (STAR) family of RNA-binding proteins, as a novel glioblastoma multiforme (GBM) tumor suppressor. Here, we establish that p53 directly regulates QKI gene expression, and QKI protein associates with and leads to the stabilization of miR-20a; miR-20a, in turn, regulates TGFßR2 and the TGFß signaling network. This pathway circuitry is substantiated by in silico epistasis analysis of its components in the human GBM TCGA (The Cancer Genome Atlas Project) collection and by their gain- and loss-of-function interactions in in vitro and in vivo complementation studies. This p53-QKI-miR-20a-TGFß pathway expands our understanding of the p53 tumor suppression network in cancer and reveals a novel tumor suppression mechanism involving regulation of specific cancer-relevant microRNAs.
Subject(s)
Cell Line , Glioblastoma/metabolism , MicroRNAs/metabolism , RNA Stability , RNA-Binding Proteins/metabolism , Animals , Glioblastoma/genetics , Humans , Mice , MicroRNAs/genetics , RNA-Binding Proteins/genetics , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Stress granules (SGs) are cytoplasmic bodies wherein translationally silenced mRNAs are recruited for triage in response to environmental stress. We report that Drosophila cells form SGs in response to arsenite and heat shock. Drosophila SGs, like mammalian SGs, are distinct from but adjacent to processing bodies (PBs, sites of mRNA silencing and decay), require polysome disassembly, and are in dynamic equilibrium with polysomes. We further examine the role of the two Drosophila eIF2alpha kinases, PEK and GCN2, in regulating SG formation in response to heat and arsenite stress. While arsenite-induced SGs are dependent upon eIF2alpha phosphorylation, primarily via PEK, heat-induced SGs are phospho-eIF2alpha-independent. In contrast, heat-induced SGs require eIF2alpha phosphorylation in mammalian cells, as non-phosphorylatable eIF2alpha Ser51Ala mutant murine embryonic fibroblasts do not form SGs even after severe heat shock. These results suggest that mammals evolved alternative mechanisms for dealing with thermal stress.
Subject(s)
Eukaryotic Initiation Factor-2/physiology , Animals , Blotting, Western , Cell Line , Cytoplasmic Granules/metabolism , Drosophila , Eukaryotic Initiation Factor-2/metabolism , Fluorescent Antibody Technique , In Situ Hybridization , Phosphorylation , Polymerase Chain Reaction , RNA Interference , RNA, Messenger/geneticsABSTRACT
Coupling of messenger RNA (mRNA) nuclear export with prior processing steps aids in the fidelity and efficiency of mRNA transport to the cytoplasm. In this study, we show that the processes of export and polyadenylation are coupled via the Drosophila melanogaster CCCH-type zinc finger protein CG6694/dZC3H3 through both physical and functional interactions. We show that depletion of dZC3H3 from S2R+ cells results in transcript hyperadenylation. Using targeted coimmunoprecipitation and liquid chromatography mass spectrometry (MS)/MS techniques, we characterize interactions of known components of the mRNA nuclear export and polyadenylation machineries with dZC3H3. Furthermore, we demonstrate the functional conservation of this factor, as depletion of its human homologue ZC3H3 by small interfering RNA results in an mRNA export defect in human cells as well. Nuclear polyadenylated (poly(A)) RNA in ZC3H3-depleted cells is sequestered in foci removed from SC35-containing speckles, indicating a shift from the normal subnuclear distribution of poly(A) RNA. Our data suggest a model wherein ZC3H3 interfaces between the polyadenylation machinery, newly poly(A) mRNAs, and factors for transcript export.
Subject(s)
Active Transport, Cell Nucleus/physiology , Carrier Proteins/metabolism , Drosophila Proteins/metabolism , RNA Transport/physiology , RNA, Messenger/metabolism , Zinc Fingers , Animals , Carrier Proteins/genetics , Cell Line , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Gene Expression Regulation , Humans , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Poly(A)-Binding Protein I/genetics , Poly(A)-Binding Protein I/metabolism , RNA, Messenger/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Eukaryotic gene expression requires export of messenger RNAs (mRNAs) from their site of transcription in the nucleus to the cytoplasm where they are translated. While mRNA export has been studied in yeast, the complexity of gene structure and cellular function in metazoan cells has likely led to increased diversification of these organisms' export pathways. Here we report the results of a genome-wide RNAi screen in which we identify 72 factors required for polyadenylated [poly-(A(+))] mRNA export from the nucleus in Drosophila cells. Using structural and functional conservation analysis of yeast and Drosophila mRNA export factors, we expose the evolutionary divergence of eukaryotic mRNA export pathways. Additionally, we demonstrate the differential export requirements of two endogenous heat-inducible transcripts--intronless heat-shock protein 70 (HSP70) and intron-containing HSP83--and identify novel export factors that participate in HSP83 mRNA splicing. We characterize several novel factors and demonstrate their participation in interactions with known components of the Drosophila export machinery. One of these factors, Drosophila melanogaster PCI domain-containing protein 2 (dmPCID2), associates with polysomes and may bridge the transition between exported messenger ribonucleoprotein particles (mRNPs) and polysomes. Our results define the global network of factors involved in Drosophila mRNA export, reveal specificity in the export requirements of different transcripts, and expose new avenues for future work in mRNA export.
Subject(s)
Drosophila/genetics , RNA Transport , RNA, Messenger/metabolism , Animals , Cell Cycle , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Genome , Models, Biological , Molecular Sequence Data , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Protein Biosynthesis , RNA Interference , RNA Splicing , Saccharomyces cerevisiae/metabolism , Sequence AlignmentABSTRACT
Duplicated genes escape gene loss by conferring a dosage benefit or evolving diverged functions. The yeast Saccharomyces cerevisiae contains many duplicated genes encoding ribosomal proteins. Prior studies have suggested that these duplicated proteins are functionally redundant and affect cellular processes in proportion to their expression. In contrast, through studies of ASH1 mRNA in yeast, we demonstrate paralog-specific requirements for the translation of localized mRNAs. Intriguingly, these paralog-specific effects are limited to a distinct subset of duplicated ribosomal proteins. Moreover, transcriptional and phenotypic profiling of cells lacking specific ribosomal proteins reveals differences between the functional roles of ribosomal protein paralogs that extend beyond effects on mRNA localization. Finally, we show that ribosomal protein paralogs exhibit differential requirements for assembly and localization. Together, our data indicate complex specialization of ribosomal proteins for specific cellular processes and support the existence of a ribosomal code.