Dissecting the energetic architecture within an RNA tertiary structural motif via high-throughput thermodynamic measurements.

Structured RNAs and RNA/protein complexes perform critical cellular functions. They often contain structurally conserved tertiary contact "motifs," whose occurrence simplifies the RNA folding landscape. Prior studies have focused on the conformational and energetic modularity of intact motifs. Here, we turn to the dissection of one common motif, the 11nt receptor (11ntR), using quantitative analysis of RNA on a massively parallel array to measure the binding of all single and double 11ntR mutants to GAAA and GUAA tetraloops, thereby probing the energetic architecture of the motif. While the 11ntR behaves as a motif, its cooperativity is not absolute. Instead, we uncovered a gradient from high cooperativity amongst base-paired and neighboring residues to additivity between distant residues. As expected, substitutions at residues in direct contact with the GAAA tetraloop resulted in the largest decreases to binding, and energetic penalties of mutations were substantially smaller for binding to the alternate GUAA tetraloop, which lacks tertiary contacts present with the canonical GAAA tetraloop. However, we found that the energetic consequences of base partner substitutions are not, in general, simply described by base pair type or isostericity. We also found exceptions to the previously established stability-abundance relationship for 11ntR sequence variants. These findings of "exceptions to the rule" highlight the power of systematic high-throughput approaches to uncover novel variants for future study in addition to providing an energetic map of a functional RNA.

Multifaceted role for p53 in pancreatic cancer suppression.

The vast majority of human pancreatic ductal adenocarcinomas (PDACs) harbor TP53 mutations, underscoring p53's critical role in PDAC suppression. PDAC can arise when pancreatic acinar cells undergo acinar-to-ductal metaplasia (ADM), giving rise to premalignant pancreatic intraepithelial neoplasias (PanINs), which finally progress to PDAC. The occurrence of TP53 mutations in late-stage PanINs has led to the idea that p53 acts to suppress malignant transformation of PanINs to PDAC. However, the cellular basis for p53 action during PDAC development has not been explored in detail. Here, we leverage a hyperactive p53 variant-p5353,54-which we previously showed is a more robust PDAC suppressor than wild-type p53, to elucidate how p53 acts at the cellular level to dampen PDAC development. Using both inflammation-induced and KRASG12D-driven PDAC models, we find that p5353,54 both limits ADM accumulation and suppresses PanIN cell proliferation and does so more effectively than wild-type p53. Moreover, p5353,54 suppresses KRAS signaling in PanINs and limits effects on the extracellular matrix (ECM) remodeling. While p5353,54 has highlighted these functions, we find that pancreata in wild-type p53 mice similarly show less ADM, as well as reduced PanIN cell proliferation, KRAS signaling, and ECM remodeling relative to Trp53-null mice. We find further that p53 enhances chromatin accessibility at sites controlled by acinar cell identity transcription factors. These findings reveal that p53 acts at multiple stages to suppress PDAC, both by limiting metaplastic transformation of acini and by dampening KRAS signaling in PanINs, thus providing key new understanding of p53 function in PDAC.

A comprehensive thermodynamic model for RNA binding by the Saccharomyces cerevisiae Pumilio protein PUF4.

Genomic methods have been valuable for identifying RNA-binding proteins (RBPs) and the genes, pathways, and processes they regulate. Nevertheless, standard motif descriptions cannot be used to predict all RNA targets or test quantitative models for cellular interactions and regulation. We present a complete thermodynamic model for RNA binding to the S. cerevisiae Pumilio protein PUF4 derived from direct binding data for 6180 RNAs measured using the RNA on a massively parallel array (RNA-MaP) platform. The PUF4 model is highly similar to that of the related RBPs, human PUM2 and PUM1, with one marked exception: a single favorable site of base flipping for PUF4, such that PUF4 preferentially binds to a non-contiguous series of residues. These results are foundational for developing and testing cellular models of RNA-RBP interactions and function, for engineering RBPs, for understanding the biophysical nature of RBP binding and the evolutionary landscape of RNAs and RBPs.

High-throughput dissection of the thermodynamic and conformational properties of a ubiquitous class of RNA tertiary contact motifs.

Despite RNA's diverse secondary and tertiary structures and its complex conformational changes, nature utilizes a limited set of structural "motifs"-helices, junctions, and tertiary contact modules-to build diverse functional RNAs. Thus, in-depth descriptions of a relatively small universe of RNA motifs may lead to predictive models of RNA tertiary conformational landscapes. Motifs may have different properties depending on sequence and secondary structure, giving rise to subclasses that expand the universe of RNA building blocks. Yet we know very little about motif subclasses, given the challenges in mapping conformational properties in high throughput. Previously, we used "RNA on a massively parallel array" (RNA-MaP), a quantitative, high-throughput technique, to study thousands of helices and two-way junctions. Here, we adapt RNA-MaP to study the thermodynamic and conformational properties of tetraloop/tetraloop receptor (TL/TLR) tertiary contact motifs, analyzing 1,493 TLR sequences from different classes. Clustering analyses revealed variability in TL specificity, stability, and conformational behavior. Nevertheless, natural GAAA/11ntR TL/TLRs, while varying in tertiary stability by ∼2.5 kcal/mol, exhibited conserved TL specificity and conformational properties. Thus, RNAs may tune stability without altering the overall structure of these TL/TLRs. Furthermore, their stability correlated with natural frequency, suggesting thermodynamics as the dominant selection pressure. In contrast, other TL/TLRs displayed heterogenous conformational behavior and appear to not be under strong thermodynamic selection. Our results build toward a generalizable model of RNA-folding thermodynamics based on the properties of isolated motifs, and our characterized TL/TLR library can be used to engineer RNAs with predictable thermodynamic and conformational behavior.

Sequence-dependent RNA helix conformational preferences predictably impact tertiary structure formation.

Structured RNAs and RNA complexes underlie biological processes ranging from control of gene expression to protein translation. Approximately 50% of nucleotides within known structured RNAs are folded into Watson-Crick (WC) base pairs, and sequence changes that preserve these pairs are typically assumed to preserve higher-order RNA structure and binding of macromolecule partners. Here, we report that indirect effects of the helix sequence on RNA tertiary stability are, in fact, significant but are nevertheless predictable from a simple computational model called RNAMake-∆∆G. When tested through the RNA on a massively parallel array (RNA-MaP) experimental platform, blind predictions for >1500 variants of the tectoRNA heterodimer model system achieve high accuracy (rmsd 0.34 and 0.77 kcal/mol for sequence and length changes, respectively). Detailed comparison of predictions to experiments support a microscopic picture of how helix sequence changes subtly modulate conformational fluctuations at each base-pair step, which accumulate to impact RNA tertiary structure stability. Our study reveals a previously overlooked phenomenon in RNA structure formation and provides a framework of computation and experiment for understanding helix conformational preferences and their impact across biological RNA and RNA-protein assemblies.

A Quantitative and Predictive Model for RNA Binding by Human Pumilio Proteins.

High-throughput methodologies have enabled routine generation of RNA target sets and sequence motifs for RNA-binding proteins (RBPs). Nevertheless, quantitative approaches are needed to capture the landscape of RNA-RBP interactions responsible for cellular regulation. We have used the RNA-MaP platform to directly measure equilibrium binding for thousands of designed RNAs and to construct a predictive model for RNA recognition by the human Pumilio proteins PUM1 and PUM2. Despite prior findings of linear sequence motifs, our measurements revealed widespread residue flipping and instances of positional coupling. Application of our thermodynamic model to published in vivo crosslinking data reveals quantitative agreement between predicted affinities and in vivo occupancies. Our analyses suggest a thermodynamically driven, continuous Pumilio-binding landscape that is negligibly affected by RNA structure or kinetic factors, such as displacement by ribosomes. This work provides a quantitative foundation for dissecting the cellular behavior of RBPs and cellular features that impact their occupancies.

Linking RNA Sequence, Structure, and Function on Massively Parallel High-Throughput Sequencers.

High-throughput sequencing methods have revolutionized our ability to catalog the diversity of RNAs and RNA-protein interactions that can exist in our cells. However, the relationship between RNA sequence, structure, and function is enormously complex, demonstrating the need for methods that can provide quantitative thermodynamic and kinetic measurements of macromolecular interaction with RNA, at a scale commensurate with the sequence diversity of RNA. Here, we discuss a class of methods that extend the core functionality of DNA sequencers to enable high-throughput measurements of RNA folding and RNA-protein interactions. Topics discussed include a description of the method and multiple applications to RNA-binding proteins, riboswitch design and engineering, and RNA tertiary structure energetics.

Intertumoral Heterogeneity in SCLC Is Influenced by the Cell Type of Origin.

The extent to which early events shape tumor evolution is largely uncharacterized, even though a better understanding of these early events may help identify key vulnerabilities in advanced tumors. Here, using genetically defined mouse models of small cell lung cancer (SCLC), we uncovered distinct metastatic programs attributable to the cell type of origin. In one model, tumors gain metastatic ability through amplification of the transcription factor NFIB and a widespread increase in chromatin accessibility, whereas in the other model, tumors become metastatic in the absence of NFIB-driven chromatin alterations. Gene-expression and chromatin accessibility analyses identify distinct mechanisms as well as markers predictive of metastatic progression in both groups. Underlying the difference between the two programs was the cell type of origin of the tumors, with NFIB-independent metastases arising from mature neuroendocrine cells. Our findings underscore the importance of the identity of cell type of origin in influencing tumor evolution and metastatic mechanisms.Significance: We show that SCLC can arise from different cell types of origin, which profoundly influences the eventual genetic and epigenetic changes that enable metastatic progression. Understanding intertumoral heterogeneity in SCLC, and across cancer types, may illuminate mechanisms of tumor progression and uncover how the cell type of origin affects tumor evolution. Cancer Discov; 8(10); 1316-31. ©2018 AACR. See related commentary by Pozo et al., p. 1216 This article is highlighted in the In This Issue feature, p. 1195.

Variable chromatin structure revealed by in situ spatially correlated DNA cleavage mapping.

Chromatin structure at the length scale encompassing local nucleosome-nucleosome interactions is thought to play a crucial role in regulating transcription and access to DNA. However, this secondary structure of chromatin remains poorly understood compared with the primary structure of single nucleosomes or the tertiary structure of long-range looping interactions. Here we report the first genome-wide map of chromatin conformation in human cells at the 1-3 nucleosome (50-500 bp) scale, obtained using ionizing radiation-induced spatially correlated cleavage of DNA with sequencing (RICC-seq) to identify DNA-DNA contacts that are spatially proximal. Unbiased analysis of RICC-seq signal reveals regional enrichment of DNA fragments characteristic of alternating rather than adjacent nucleosome interactions in tri-nucleosome units, particularly in H3K9me3-marked heterochromatin. We infer differences in the likelihood of nucleosome-nucleosome contacts among open chromatin, H3K27me3-marked, and H3K9me3-marked repressed chromatin regions. After calibrating RICC-seq signal to three-dimensional distances, we show that compact two-start helical fibre structures with stacked alternating nucleosomes are consistent with RICC-seq fragmentation patterns from H3K9me3-marked chromatin, while non-compact structures and solenoid structures are consistent with open chromatin. Our data support a model of chromatin architecture in intact interphase nuclei consistent with variable longitudinal compaction of two-start helical fibres.

Nfib Promotes Metastasis through a Widespread Increase in Chromatin Accessibility.

Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.

Structured nucleosome fingerprints enable high-resolution mapping of chromatin architecture within regulatory regions.

Transcription factors canonically bind nucleosome-free DNA, making the positioning of nucleosomes within regulatory regions crucial to the regulation of gene expression. Using the assay of transposase accessible chromatin (ATAC-seq), we observe a highly structured pattern of DNA fragment lengths and positions around nucleosomes in Saccharomyces cerevisiae, and use this distinctive two-dimensional nucleosomal "fingerprint" as the basis for a new nucleosome-positioning algorithm called NucleoATAC. We show that NucleoATAC can identify the rotational and translational positions of nucleosomes with up to base-pair resolution and provide quantitative measures of nucleosome occupancy in S. cerevisiae, Schizosaccharomyces pombe, and human cells. We demonstrate the application of NucleoATAC to a number of outstanding problems in chromatin biology, including analysis of sequence features underlying nucleosome positioning, promoter chromatin architecture across species, identification of transient changes in nucleosome occupancy and positioning during a dynamic cellular response, and integrated analysis of nucleosome occupancy and transcription factor binding.