Rapid, robust plasmid verification by de novo assembly of short sequencing reads.

Plasmids are a foundational tool for basic and applied research across all subfields of biology. Increasingly, researchers in synthetic biology are relying on and developing massive libraries of plasmids as vectors for directed evolution, combinatorial gene circuit tests, and for CRISPR multiplexing. Verification of plasmid sequences following synthesis is a crucial quality control step that creates a bottleneck in plasmid fabrication workflows. Crucially, researchers often elect to forego the cumbersome verification step, potentially leading to reproducibility and-depending on the application-security issues. In order to facilitate plasmid verification to improve the quality and reproducibility of life science research, we developed a fast, simple, and open source pipeline for assembly and verification of plasmid sequences from Illumina reads. We demonstrate that our pipeline, which relies on de novo assembly, can also be used to detect contaminating sequences in plasmid samples. In addition to presenting our pipeline, we discuss the role for verification and quality control in the increasingly complex life science workflows ushered in by synthetic biology.

Imaging translational control by Argonaute with single-molecule resolution in live cells.

A major challenge to our understanding of translational control has been deconvolving the individual impact specific regulatory factors have on the complex dynamics of mRNA translation. MicroRNAs (miRNAs), for example, guide Argonaute and associated proteins to target mRNAs, where they direct gene silencing in multiple ways that are not well understood. To better deconvolve these dynamics, we have developed technology to directly visualize and quantify the impact of human Argonaute2 (Ago2) on the translation and subcellular localization of individual reporter mRNAs in living cells. We show that our combined translation and Ago2 tethering sensor reflects endogenous miRNA-mediated gene silencing. Using the sensor, we find that Ago2 association leads to progressive silencing of translation at individual mRNA. Silencing was occasionally interrupted by brief bursts of translational activity and took 3-4 times longer than a single round of translation, consistent with a gradual increase in the inhibition of translation initiation. At later time points, Ago2-tethered mRNAs cluster and coalesce with P-bodies, where a translationally silent state is maintained. These results provide a framework for exploring miRNA-mediated gene regulation in live cells at the single-molecule level. Furthermore, our tethering-based, single-molecule reporter system will likely have wide-ranging application in studying RNA-protein interactions.

Dual roles for piRNAs in promoting and preventing gene silencing in C. elegans.

Piwi-interacting RNAs (piRNAs) regulate many biological processes through mechanisms that are not fully understood. In Caenorhabditis elegans, piRNAs intersect the endogenous RNA interference (RNAi) pathway, involving a distinct class of small RNAs called 22G-RNAs, to regulate gene expression in the germline. In the absence of piRNAs, 22G-RNA production from many genes is reduced, pointing to a role for piRNAs in facilitating endogenous RNAi. Here, however, we show that many genes gain, rather than lose, 22G-RNAs in the absence of piRNAs, which is in some instances coincident with RNA silencing. Aberrant 22G-RNA production is somewhat stochastic but once established can occur within a population for at least 50 generations. Thus, piRNAs both promote and suppress 22G-RNA production and gene silencing. rRNAs and histones are hypersusceptible to aberrant silencing, but we do not find evidence that their misexpression is the primary cause of the transgenerational sterility observed in piRNA-defective mutants.

Lighting up single-mRNA translation dynamics in living cells.

Over the past five years, technological advances have made it possible to image the translation of single mRNA in the natural context of living cells. With these advances, researchers are beginning to shed light on when, where, and to what degree mRNA are translated with single-molecule precision. These works provide insight into the heterogeneity of translation amongst single transcripts, behavior that is averaged out in complementary bulk assays. In this review, we discuss the rapidly maturing field of live-cell, single-mRNA imaging of translation, beginning with a brief overview of recent technological advances. The remainder of the review focuses on the new biological insights gained from these technologies. We conclude with a discussion of the future of this technology.

Castration Determines the Efficacy of ETAR Blockade in a Mouse Model of Prostate Cancer Bone Metastasis.

Bone metastasis is a painful complication of advanced prostate cancer. Endothelin-1 is a tumor-secreted factor that plays a central role in osteoblast activation and the osteosclerotic response of prostate cancer metastatic to bone. Antagonists that block the activation of the endothelin A receptor (ETAR), located on osteoblasts, reduce osteoblastic bone lesions in animal models of bone metastasis. However, ETAR antagonists demonstrated limited efficacy in clinical trials of men with advanced prostate cancer who also received standard androgen deprivation therapy (ADT). Previous data from our group suggested that, in a mouse model, ETAR antagonists might only be efficacious when androgen signaling in the osteoblast is lowered beyond the ability of standard ADT. This notion was tested in a mouse model of prostate cancer bone metastasis. Castrated and sham-operated male athymic nude mice underwent intracardiac inoculation of the ARCaPM castration-resistant prostate cancer cell line. The mice were then treated with either the ETAR antagonist zibotentan or a vehicle control to generate four experimental groups: vehicle+sham (Veh+Sham), vehicle+castrate (Veh+Castr), zibotentan+sham (Zibo+Sham), and zibotentan+castrate (Zibo+Castr). The mice were monitored radiographically for the development of skeletal lesions. The Zibo+Castr group had significantly longer survival and a single incidental lesion. Mice in the Zibo+Sham group had the shortest survival and the largest number of skeletal lesions. Survival and skeletal lesions of the Veh+Sham and Veh+Castr groups were intermediate compared with the zibotentan-treated groups. We report a complex interaction between ETAR and androgen signaling, whereby ETAR blockade was most efficacious when combined with complete androgen deprivation.