A synthetic library of RNA control modules for predictable tuning of gene expression in yeast

Advances in synthetic biology have resulted in the development of genetic tools that support the design of complex biological systems encoding desired functions. The majority of efforts have focused on the development of regulatory tools in bacteria, whereas fewer tools exist for the tuning of expression levels in eukaryotic organisms. Here, we describe a novel class of RNA-based control modules that provide predictable tuning of expression levels in the yeast Saccharomyces cerevisiae. A library of synthetic control modules that act through posttranscriptional RNase cleavage mechanisms was generated through an in vivo screen, in which structural engineering methods were applied to enhance the insulation and modularity of the resulting components. This new class of control elements can be combined with any promoter to support titration of regulatory strategies encoded in transcriptional regulators and thus more sophisticated control schemes. We applied these synthetic controllers to the systematic titration of flux through the ergosterol biosynthesis pathway, providing insight into endogenous control strategies and highlighting the utility of this control module library for manipulating and probing biological systems

Heterogeneity in HIV and cellular transcription profiles in cell line models of latent and productive infection: implications for HIV latency.

BackgroundHIV-infected cell lines are widely used to study latent HIV infection, which is considered the main barrier to HIV cure. We hypothesized that these cell lines differ from each other and from cells from HIV-infected individuals in the mechanisms underlying latency.ResultsTo quantify the degree to which HIV expression is inhibited by blocks at different stages of HIV transcription, we employed a recently-described panel of RT-ddPCR assays to measure levels of 7 HIV transcripts ("read-through," initiated, 5' elongated, mid-transcribed/unspliced [Pol], distal-transcribed [Nef], polyadenylated, and multiply-sliced [Tat-Rev]) in bulk populations of latently-infected (U1, ACH-2, J-Lat) and productively-infected (8E5, activated J-Lat) cell lines. To assess single-cell variation and investigate cellular genes associated with HIV transcriptional blocks, we developed a novel multiplex qPCR panel and quantified single cell levels of 7 HIV targets and 89 cellular transcripts in latently- and productively-infected cell lines. The bulk cell HIV transcription profile differed dramatically between cell lines and cells from ART-suppressed individuals. Compared to cells from ART-suppressed individuals, latent cell lines showed lower levels of HIV transcriptional initiation and higher levels of polyadenylation and splicing. ACH-2 and J-Lat cells showed different forms of transcriptional interference, while U1 cells showed a block to elongation. Single-cell studies revealed marked variation between/within cell lines in expression of HIV transcripts, T cell phenotypic markers, antiviral factors, and genes implicated in latency. Expression of multiply-spliced HIV Tat-Rev was associated with expression of cellular genes involved in activation, tissue retention, T cell transcription, and apoptosis/survival.ConclusionsHIV-infected cell lines differ from each other and from cells from ART-treated individuals in the mechanisms governing latent HIV infection. These differences in viral and cellular gene expression must be considered when gauging the suitability of a given cell line for future research on HIV. At the same time, some features were shared across cell lines, such as low expression of antiviral defense genes and a relationship between productive infection and genes involved in survival. These features may contribute to HIV latency or persistence in vivo, and deserve further study using novel single cell assays such as those described in this manuscript

Detection of Plasmodium falciparum male and female gametocytes and determination of parasite sex ratio in human endemic populations by novel, cheap and robust RTqPCR assays

The presence of Plasmodium falciparum gametocytes in peripheral blood is essential for human to mosquito parasite transmission. The detection of submicroscopic infections with gametocytes and the estimation of the gametocyte sex ratio are crucial to assess the human host potential ability to infect mosquitoes and transmit malaria parasites

Synthetic RNA modules for fine-tuning gene expression levels in yeast by modulating RNase III activity

The design of synthetic gene networks requires an extensive genetic toolbox to control the activities and levels of protein components to achieve desired cellular functions. Recently, a novel class of RNA-based control modules, which act through post-transcriptional processing of transcripts by directed RNase III (Rnt1p) cleavage, were shown to provide predictable control over gene expression and unique properties for manipulating biological networks. Here, we increase the regulatory range of the Rnt1p control elements, by modifying a critical region for enzyme binding to its hairpin substrates, the binding stability box (BSB). We used a high throughput, cell-based selection strategy to screen a BSB library for sequences that exhibit low fluorescence and thus high Rnt1p processing efficiencies. Sixteen unique BSBs were identified that cover a range of protein expression levels, due to the ability of the sequences to affect the hairpin cleavage rate and to form active cleavable complexes with Rnt1p. We further demonstrated that the activity of synthetic Rnt1p hairpins can be rationally programmed by combining the synthetic BSBs with a set of sequences located within a different region of the hairpin that directly modulate cleavage rates, providing a modular assembly strategy for this class of RNA-based control elements

Synthetic control of a fitness tradeoff in yeast nitrogen metabolism

Background: Microbial communities are involved in many processes relevant to industrial and medical biotechnology, such as the formation of biofilms, lignocellulosic degradation, and hydrogen production. The manipulation of synthetic and natural microbial communities and their underlying ecological parameters, such as fitness, evolvability, and variation, is an increasingly important area of research for synthetic biology. Results: Here, we explored how synthetic control of an endogenous circuit can be used to regulate a tradeoff between fitness in resource abundant and resource limited environments in a population of Saccharomyces cerevisiae. We found that noise in the expression of a key enzyme in ammonia assimilation, Gdh1p, mediated a tradeoff between growth in low nitrogen environments and stress resistance in high ammonia environments. We implemented synthetic control of an endogenous Gdh1p regulatory network to construct an engineered strain in which the fitness of the population was tunable in response to an exogenously-added small molecule across a range of ammonia environments. Conclusion: The ability to tune fitness and biological tradeoffs will be important components of future efforts to engineer microbial communities

Up-regulation of microRNA-21 correlates with lower kidney cancer survival.

BackgroundMicroRNA-21 is up-regulated in a variety of cancers like, breast, colorectal, lung, head and neck etc. However, the regulation of miR-21 in renal cell carcinoma (RCC) has not yet been studied systematically.Methods and resultsWe measured miR-21 levels in 54 pairs of kidney cancers and their normal matched tissues by real-time PCR. The expression level of miR-21 was correlated with 5 year survival and the pathological stage. Functional studies were done after inhibiting miR-21 in RCC cell lines. We studied in vitro and in vivo effects of the chemo preventive agent genistein on miR-21 expression. In 48 cases (90%), miR-21 was increased. All patients with low miR-21 expression survived 5 years, while with high miR-21 expression, only 50% survived. Higher expression of miR-21 is associated with an increase in the stage of renal cancer. Functional studies after inhibiting miRNA-21 in RCC cell lines show cell cycle arrest, induction of apoptosis and reduced invasive and migratory capabilities. Western blot analysis showed an increase in the expression of p21 and p38 MAP kinase genes and a reduction in cyclin E2. Genistein inhibited the expression of miR-21 in A-498 cells and in the tumors formed after injecting genistein treated A-498 cells in nude mice besides inhibiting tumor formation.ConclusionsThe current study shows a clear correlation between miR-21 expression and clinical characteristics of renal cancer. Thus we believe that miR-21 can be used as a tumor marker and its inhibition may prove to be useful in controlling cancers with up-regulated miR-21

Global landscape of mouse and human cytokine transcriptional regulation

Cytokines are cell-to-cell signaling proteins that play a central role in immune development, pathogen responses, and diseases. Cytokines are highly regulated at the transcriptional level by combinations of transcription factors (TFs) that recruit cofactors and the transcriptional machinery. Here, we mined through three decades of studies to generate a comprehensive database, CytReg, reporting 843 and 647 interactions between TFs and cytokine genes, in human and mouse respectively. By integrating CytReg with other functional datasets, we determined general principles governing the transcriptional regulation of cytokine genes. In particular, we show a correlation between TF connectivity and immune phenotype and disease, we discuss the balance between tissue-specific and pathogen-activated TFs regulating each cytokine gene, and cooperativity and plasticity in cytokine regulation. We also illustrate the use of our database as a blueprint to predict TF–disease associations and identify potential TF–cytokine regulatory axes in autoimmune diseases. Finally, we discuss research biases in cytokine regulation studies, and use CytReg to predict novel interactions based on co-expression and motif analyses which we further validated experimentally. Overall, this resource provides a framework for the rational design of future cytokine gene regulation studies.National Institutes of Health (NIH) [R00 GM114296 and R35 GM128625 to J.I.F.B., 5T32HL007501-34 to J.A.S.]; National Science Foundation [NSF-REU BIO-1659605 to M.M.]. Funding for open access charge: NIH [R35 GM128625]. (R00 GM114296 - National Institutes of Health (NIH); R35 GM128625 - National Institutes of Health (NIH); 5T32HL007501-34 - National Institutes of Health (NIH); NSF-REU BIO-1659605 - National Science Foundation; R35 GM128625 - NIH)Published versio