A Conserved CCCH-type Zinc Finger Protein Regulates mRNA Nuclear Adenylation and Export

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

Comparative analysis of three studies measuring fluorescence from engineered bacterial genetic constructs

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

Robust estimation of bacterial cell count from optical density

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

Synthetic Biology for Global Good: Volume I

This collection of articles represents the outcomes of our approach to engaging students as direct contributors to the synthetic biology primary literature. BB4260:Synthetic Biology is a seven-week, three-credit course, and uses a medley of mini-lectures, case studies, discussion, and active learning to explore current primary literature in the field. Thirty-one students majoring in life sciences (biology and biotechnology, biochemistry, or biomedical engineering), primarily juniors and seniors, enrolled in and completed the course. As part of this course, we created a project designed to engage students deeply in the process of creating biological literature by writing a mini-review style article, and engaging in the process of peer review. To define the theme of their article, and to help students place their projects within the context of important global challenges, we prompted each student to review the 17 Sustainable Development Goals identified by the United Nations and rank the goals according to their personal interests. We then used these rankings to create five project groups of students with like interests. From there, students searched the primary literature to refine their topic ideas within their groups. The students iterated on their projects by writing and revising a total of three drafts. The student authors have made important contributions in the areas of human well-being, equity, sustainability, and environmental health, through their insightful synthesis of the literature into the following articles in this collection: “Carbon Capture by Transgenic Plant Greenspaces in Urban Communities” by Katherine Stratton, Hannah Shell, Anna Wix, William Miller, Hayley Wigren, and Priscilla Anand; “Water Quality Biosensing Using Engineered Microbial Fuel Cells” by Erik Breiling, John Gabelmann, Rachel Grandmaison, Caitlin Guifoyle, Taylor Johnson, and Adam LaBombard; “Improving Inequalities: Protein-Based vs. mRNA Vaccines” by Shelby Tweedie, Maire Murphy, Bethany Atwood, Jocelyn Hinchcliffe, Samantha Lopez, and Andrew Voronin; “Hot & Bothered: Engineered Microbes to Treat GI Inflammation” by Hope Hutchinson, Lauren Abraham, Alyssa Carta, Gabrielle Paquette, Kaitlyn Bergeron, and Kaleigh Caserta; “Enhancing Algae Biomass from Biofuel Production as an Alternative Feed for Livestock” by Gianluca Panza, Mira Kirschner, Aashi Akare, Dylan Mackisey, Mikayla Raffin, and Komlavi Touglo. Generating and disseminating this collection was made possible via the generous support of the WPI Women’s Impact Network (WIN) EmpOwER program

Unlocking the strength of inducible promoters in Gram‐negative bacteria

Abstract 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

Definition of global and transcript-specific mRNA export pathways in metazoans

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

Functional Specificity among Ribosomal Proteins Regulates Gene Expression

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