Causes, functions, and therapeutic possibilities of RNA secondary structure ensembles and alternative states.

RNA secondary structure plays essential roles in encoding RNA regulatory fate and function. Most RNAs populate ensembles of alternatively paired states and are continually unfolded and refolded by cellular processes. Measuring these structural ensembles and their contributions to cellular function has traditionally posed major challenges, but new methods and conceptual frameworks are beginning to fill this void. In this review, we provide a mechanism- and function-centric compendium of the roles of RNA secondary structural ensembles and minority states in regulating the RNA life cycle, from transcription to degradation. We further explore how dysregulation of RNA structural ensembles contributes to human disease and discuss the potential of drugging alternative RNA states to therapeutically modulate RNA activity. The emerging paradigm of RNA structural ensembles as central to RNA function provides a foundation for a deeper understanding of RNA biology and new therapeutic possibilities.

Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS.

Chemical probing experiments have transformed RNA structure analysis, enabling high-throughput measurement of base-pairing in living cells. Dimethyl sulfate (DMS) is one of the most widely used structure probing reagents and has played a pivotal role in enabling next-generation single-molecule probing analyses. However, DMS has traditionally only been able to probe adenine and cytosine nucleobases. We previously showed that, using appropriate conditions, DMS can also be used to interrogate base-pairing of uracil and guanines in vitro at reduced accuracy. However, DMS remained unable to informatively probe guanines in cells. Here, we develop an improved DMS mutational profiling (MaP) strategy that leverages the unique mutational signature of N1-methylguanine DMS modifications to enable high-fidelity structure probing at all four nucleotides, including in cells. Using information theory, we show that four-base DMS reactivities convey greater structural information than current two-base DMS and SHAPE probing strategies. Four-base DMS experiments further enable improved direct base-pair detection by single-molecule PAIR analysis, and ultimately support RNA structure modeling at superior accuracy. Four-base DMS probing experiments are straightforward to perform and will broadly facilitate improved RNA structural analysis in living cells.

Mutation signature filtering enables high-fidelity RNA structure probing at all four nucleobases with DMS.

Chemical probing experiments have transformed RNA structure analysis, enabling high-throughput measurement of base-pairing in living cells. Dimethyl sulfate (DMS) is one of the most widely used structure probing reagents and has played a prominent role in enabling next-generation single-molecule probing analyses. However, DMS has traditionally only been able to probe adenine and cytosine nucleobases. We previously showed that, using appropriate conditions, DMS can also be used to interrogate base-pairing of uracil and guanines in vitro at reduced accuracy. However, DMS remained unable to informatively probe guanines in cells. Here, we develop an improved DMS mutational profiling (MaP) strategy that leverages the unique mutational signature of N 1 -methylguanine DMS modifications to enable robust, high-fidelity structure probing at all four nucleotides, including in cells. Using information theory, we show that four-base DMS reactivities convey greater structural information than comparable two-base DMS and SHAPE probing strategies. Four-base DMS experiments further enable improved direct base-pair detection by single-molecule PAIR analysis, and ultimately support RNA structure modeling at superior accuracy. Four-base DMS probing experiments are easily performed and will broadly facilitate improved RNA structural analysis in living cells.

Ecdysoneless Overexpression Drives Mammary Tumorigenesis through Upregulation of C-MYC and Glucose Metabolism.

Ecdysoneless (ECD) protein is essential for embryogenesis, cell-cycle progression, and cellular stress mitigation with an emerging role in mRNA biogenesis. We have previously shown that ECD protein as well as its mRNA are overexpressed in breast cancer and ECD overexpression predicts shorter survival in patients with breast cancer. However, the genetic evidence for an oncogenic role of ECD has not been established. Here, we generated transgenic mice with mammary epithelium-targeted overexpression of an inducible human ECD transgene (ECDTg). Significantly, ECDTg mice develop mammary hyperplasia, preneoplastic lesions, and heterogeneous tumors with occasional lung metastasis. ECDTg tumors exhibit epithelial to mesenchymal transition and cancer stem cell characteristics. Organoid cultures of ECDTg tumors showed ECD dependency for in vitro oncogenic phenotype and in vivo growth when implanted in mice. RNA sequencing (RNA-seq) analysis of ECDTg tumors showed a c-MYC signature, and alterations in ECD levels regulated c-MYC mRNA and protein levels as well as glucose metabolism. ECD knockdown-induced decrease in glucose uptake was rescued by overexpression of mouse ECD as well as c-MYC. Publicly available expression data analyses showed a significant correlation of ECD and c-MYC overexpression in breast cancer, and ECD and c-MYC coexpression exhibits worse survival in patients with breast cancer. Taken together, we establish a novel role of overexpressed ECD as an oncogenesis driver in the mouse mammary gland through upregulation of c-MYC-mediated glucose metabolism. IMPLICATIONS: We demonstrate ECD overexpression in the mammary gland of mice led to the development of a tumor progression model through upregulation of c-MYC signaling and glucose metabolism.

Discovery of a large-scale, cell-state-responsive allosteric switch in the 7SK RNA using DANCE-MaP.

7SK is a conserved noncoding RNA that regulates transcription by sequestering the transcription factor P-TEFb. 7SK function entails complex changes in RNA structure, but characterizing RNA dynamics in cells remains an unsolved challenge. We developed a single-molecule chemical probing strategy, DANCE-MaP (deconvolution and annotation of ribonucleic conformational ensembles), that defines per-nucleotide reactivity, direct base pairing interactions, tertiary interactions, and thermodynamic populations for each state in RNA structural ensembles from a single experiment. DANCE-MaP reveals that 7SK RNA encodes a large-scale structural switch that couples dissolution of the P-TEFb binding site to structural remodeling at distal release factor binding sites. The 7SK structural equilibrium shifts in response to cell growth and stress and can be targeted to modulate expression of P-TEFbresponsive genes. Our study reveals that RNA structural dynamics underlie 7SK function as an integrator of diverse cellular signals to control transcription and establishes the power of DANCE-MaP to define RNA dynamics in cells.

Ecdysoneless Protein Regulates Viral and Cellular mRNA Splicing to Promote Cervical Oncogenesis.

High-risk human papillomaviruses (HPV), exemplified by HPV16/18, are causally linked to human cancers of the anogenital tract, skin, and upper aerodigestive tract. Previously, we identified Ecdysoneless (ECD) protein, the human homolog of the Drosophila ecdysoneless gene, as a novel HPV16 E6-interacting protein. Here, we show that ECD, through its C-terminal region, selectively binds to high-risk but not to low-risk HPV E6 proteins. We demonstrate that ECD is overexpressed in cervical and head and neck squamous cell carcinoma (HNSCC) cell lines as well as in tumor tissues. Using The Cancer Genome Atlas dataset, we show that ECD mRNA overexpression predicts shorter survival in patients with cervical and HNSCC. We demonstrate that ECD knockdown in cervical cancer cell lines led to impaired oncogenic behavior, and ECD co-overexpression with E7 immortalized primary human keratinocytes. RNA-sequencing analyses of SiHa cells upon ECD knockdown showed to aberrations in E6/E7 RNA splicing, as well as RNA splicing of several HPV oncogenesis-linked cellular genes, including splicing of components of mRNA splicing machinery itself. Taken together, our results support a novel role of ECD in viral and cellular mRNA splicing to support HPV-driven oncogenesis. IMPLICATIONS: This study links ECD overexpression to poor prognosis and shorter survival in HNSCC and cervical cancers and identifies a critical role of ECD in cervical oncogenesis through regulation of viral and cellular mRNA splicing.

The Mammalian Ecdysoneless Protein Interacts with RNA Helicase DDX39A To Regulate Nuclear mRNA Export.

The mammalian orthologue of ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of endoplasmic reticulum stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. We find that ECD is involved in RNA export through its interaction with DDX39A. ECD knockdown (KD) blocks mRNA export from the nucleus to the cytoplasm, which is rescued by expression of full-length ECD but not an ECD mutant that is defective in interaction with DDX39A. We have previously shown that ECD protein is overexpressed in ErbB2+ breast cancers (BC). In this study, we extended the analyses to two publicly available BC mRNA The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) data sets. In both data sets, ECD mRNA overexpression correlated with short patient survival, specifically ErbB2+ BC. In the METABRIC data set, ECD overexpression also correlated with poor patient survival in triple-negative breast cancer (TNBC). Furthermore, ECD KD in ErbB2+ BC cells led to a decrease in ErbB2 mRNA level due to a block in its nuclear export and was associated with impairment of oncogenic traits. These findings provide novel mechanistic insight into the physiological and pathological functions of ECD.

Mammalian ECD Protein Is a Novel Negative Regulator of the PERK Arm of the Unfolded Protein Response.

Mammalian Ecdysoneless (ECD) is a highly conserved ortholog of the DrosophilaEcd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling.