Tuning strand displacement kinetics enables programmable ZTP riboswitch dynamic range in vivo.

A large body of work has shown that transcriptional riboswitches function through internal strand displacement mechanisms that guide the formation of alternative structures which drive regulatory outcomes. Here, we sought to investigate this phenomenon using the Clostridium beijerinckii pfl ZTP riboswitch as a model system. Using functional mutagenesis with Escherichia coli gene expression assays, we show that mutations designed to slow strand displacement of the expression platform enable precise tuning of riboswitch dynamic range (2.4-34-fold), depending on the type of kinetic barrier introduced, and the position of the barrier relative to the strand displacement nucleation site. We also show that expression platforms from a range of different Clostridium ZTP riboswitches contain sequences that impose these barriers to affect dynamic range in these different contexts. Finally, we use sequence design to flip the regulatory logic of the riboswitch to create a transcriptional OFF-switch, and show that the same barriers to strand displacement tune dynamic range in this synthetic context. Together, our findings further elucidate how strand displacement can be manipulated to alter the riboswitch decision landscape, suggesting that this could be a mechanism by which evolution tunes riboswitch sequence, and providing an approach to optimize synthetic riboswitches for biotechnology applications.

RNA folding kinetics control riboswitch sensitivity in vivo.

Riboswitches are ligand-responsive gene-regulatory RNA elements that perform key roles in maintaining cellular homeostasis. Understanding how riboswitch sensitivity is controlled is critical to understanding how highly conserved aptamer domains are deployed in a variety of contexts with different sensitivity demands. Here we uncover new roles by which RNA folding dynamics control riboswitch sensitivity in cells. By investigating the Clostridium beijerinckii pfl ZTP riboswitch, we identify multiple mechanistic routes of altering expression platform sequence and structure to slow RNA folding, all of which enhance riboswitch sensitivity. Applying these methods to riboswitches with diverse aptamer architectures that regulate transcription and translation with ON and OFF logic demonstrates the generality of our findings, indicating that any riboswitch that operates in a kinetic regime can be sensitized by slowing expression platform folding. Comparison of the most sensitized versions of these switches to equilibrium aptamer:ligand dissociation constants suggests a limit to the sensitivities achievable by kinetic RNA switches. Our results add to the growing suite of knowledge and approaches that can be used to rationally program cotranscriptional RNA folding for biotechnology applications, and suggest general RNA folding principles for understanding dynamic RNA systems in other areas of biology.

How does RNA fold dynamically?

Recent advances in interrogating RNA folding dynamics have shown the classical model of RNA folding to be incomplete. Here, we pose three prominent questions for the field that are at the forefront of our understanding of the importance of RNA folding dynamics for RNA function. The first centers on the most appropriate biophysical framework to describe changes to the RNA folding energy landscape that a growing RNA chain encounters during transcriptional elongation. The second focuses on the potential ubiquity of strand displacement - a process by which RNA can rapidly change conformations - and how this process may be generally present in broad classes of seemingly different RNAs. The third raises questions about the potential importance and roles of cellular protein factors in RNA conformational switching. Answers to these questions will greatly improve our fundamental knowledge of RNA folding and function, drive biotechnological advances that utilize engineered RNAs, and potentially point to new areas of biology yet to be discovered.

RNA Engineering for Public Health: Innovations in RNA-Based Diagnostics and Therapeutics.

RNA is essential for cellular function: From sensing intra- and extracellular signals to controlling gene expression, RNA mediates a diverse and expansive list of molecular processes. A long-standing goal of synthetic biology has been to develop RNA engineering principles that can be used to harness and reprogram these RNA-mediated processes to engineer biological systems to solve pressing global challenges. Recent advances in the field of RNA engineering are bringing this to fruition, enabling the creation of RNA-based tools to combat some of the most urgent public health crises. Specifically, new diagnostics using engineered RNAs are able to detect both pathogens and chemicals while generating an easily detectable fluorescent signal as an indicator. New classes of vaccines and therapeutics are also using engineered RNAs to target a wide range of genetic and pathogenic diseases. Here, we discuss the recent breakthroughs in RNA engineering enabling these innovations and examine how advances in RNA design promise to accelerate the impact of engineered RNA systems.

Generation of Functional Gene Knockout Melanoma Cell Lines by CRISPR-Cas9 Gene Editing.

Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular levels. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.

RNA Sequence and Structure Determinants of Pol III Transcriptional Termination in Human Cells.

The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that multiple uracils at the end of RNA transcripts are required for Pol III termination, the effects of upstream RNA secondary structure in the nascent transcript on transcriptional termination is still unclear. To address this, we developed an in cellulo Pol III transcription termination assay using the recently developed Tornado-Corn RNA aptamer system to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. For transcription from Pol III type 3 promoters, we found that only poly-U tracts longer than the average length found in the human genome efficiently terminate Pol III transcription without RNA secondary structure elements. We observed that RNA secondary structure elements placed in proximity to shorter poly-U tracts induced termination, and RNA secondary structure by itself was not sufficient to induce termination. For Pol III type 2 promoters, we found that the shorter poly-U tract lengths of 4 uracils were sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.

The FOXC2 Transcription Factor Promotes Melanoma Outgrowth and Regulates Expression of Genes Associated With Drug Resistance and Interferon Responsiveness.

BACKGROUND/AIM: The FOXC2 transcription factor promotes the progression of several cancer types, but has not been investigated in the context of melanoma cells. To study FOXC2's influence on melanoma progression, we generated a FOXC2-deficient murine melanoma cell line and evaluated The Cancer Genome Atlas (TCGA) patient datasets. MATERIALS AND METHODS: We compared tumor growth kinetics and RNA-seq/qRT-PCR gene expression profiles from wild-type versus FOXC2-deficient murine melanomas. We also performed Kaplan-Meier survival analysis of TCGA data to assess the influence of FOXC2 gene expression on melanoma patients' response to chemotherapy and immunotherapy. RESULTS: FOXC2 promotes melanoma progression and regulates the expression of genes associated with multiple oncogenic pathways, including the oxidative stress response, xenobiotic metabolism, and interferon responsiveness. FOXC2 expression in melanoma correlates negatively with patient response to chemotherapy and immunotherapy. CONCLUSION: FOXC2 drives a tumor-promoting gene expression program in melanoma and is a prognostic indicator of patient response to multiple cancer therapies.