Riboswitch Distribution in the Human Gut Microbiome Reveals Common Metabolite Pathways.

Riboswitches are widely distributed, conserved RNAs which regulate metabolite levels in bacterial cells through direct, noncovalent binding of their cognate metabolite. Various riboswitch families are highly enriched in gut bacteria, suggestive of a symbiotic relationship between the host and bacteria. Previous studies of the distribution of riboswitches have examined bacterial taxa broadly. Thus, the distribution of riboswitches associated with bacteria inhabiting the intestines of healthy individuals is not well understood. To address these questions, we survey the gut microbiome for riboswitches by including an international database of prokaryotic genomes from the gut samples. Using Infernal, a program that uses RNA-specific sequence and structural features, we survey this data set using existing riboswitch models. We identify 22 classes of riboswitches with vitamin cofactors making up the majority of riboswitch-associated pathways. Our finding is reproducible in other representative databases from the oral as well as the marine microbiomes, underscoring the importance of thiamine pyrophosphate, cobalamin, and flavin mononucleotide in gene regulation. Interestingly, riboswitches do not vary significantly across microbiome representatives from around the world despite major taxonomic differences; this suggests an underlying conservation. Further studies elucidating the role of bacterial riboswitches in the host metabolome are needed to illuminate the consequences of our finding.

Abolished frameshifting for predicted structure-stabilizing SARS-CoV-2 mutants: Implications to alternative conformations and their statistical structural analyses.

The SARS-CoV-2 frameshifting element (FSE) has been intensely studied and explored as a therapeutic target for coronavirus diseases including COVID-19. Besides the intriguing virology, this small RNA is known to adopt many length-dependent conformations, as verified by multiple experimental and computational approaches. However, the role these alternative conformations play in the frameshifting mechanism and how to quantify this structural abundance has been an ongoing challenge. Here, we show by DMS and dual-luciferase functional assays that previously predicted FSE mutants (using the RAG graph theory approach) suppress structural transitions and abolish frameshifting. Furthermore, correlated mutation analysis of DMS data by three programs (DREEM, DRACO, and DANCE-MaP) reveals important differences in their estimation of specific RNA conformations, suggesting caution in the interpretation of such complex conformational landscapes. Overall, the abolished frameshifting in three different mutants confirms that all alternative conformations play a role in the pathways of ribosomal transition.

Regulation of chromatin architecture by transcription factor binding.

Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome to control the transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers at nucleosome resolution to determine TF effect on chromatin architecture and shed light into its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by TF binding. The position of TF binding dictates the formation of microdomains that appear visible only at the ensemble level. At the same time, the level of linker histone and tail acetylation, or different linker DNA lengths, regulates the concentration-dependent effect of TF binding. Furthermore, we show how TF binding can repress gene expression by increasing fiber folding motifs that help compact and occlude the promoter region. Importantly, this effect can be reversed by increasing linker histone density. Overall, these results shed light on the epigenetic control of the genome dictated by TF binding.

Hi-BDiSCO: folding 3D mesoscale genome structures from Hi-C data using brownian dynamics.

The structure and dynamics of the eukaryotic genome are intimately linked to gene regulation and transcriptional activity. Many chromosome conformation capture experiments like Hi-C have been developed to detect genome-wide contact frequencies and quantify loop/compartment structures for different cellular contexts and time-dependent processes. However, a full understanding of these events requires explicit descriptions of representative chromatin and chromosome configurations. With the exponentially growing amount of data from Hi-C experiments, many methods for deriving 3D structures from contact frequency data have been developed. Yet, most reconstruction methods use polymer models with low resolution to predict overall genome structure. Here we present a Brownian Dynamics (BD) approach termed Hi-BDiSCO for producing 3D genome structures from Hi-C and Micro-C data using our mesoscale-resolution chromatin model based on the Discrete Surface Charge Optimization (DiSCO) model. Our approach integrates reconstruction with chromatin simulations at nucleosome resolution with appropriate biophysical parameters. Following a description of our protocol, we present applications to the NXN, HOXC, HOXA and Fbn2 mouse genes ranging in size from 50 to 100 kb. Such nucleosome-resolution genome structures pave the way for pursuing many biomedical applications related to the epigenomic regulation of chromatin and control of human disease.

Regulation of Chromatin Architecture by Transcription Factor Binding.

Transcription factors (TF) bind to chromatin and regulate the expression of genes. The pair Myc:Max binds to E-box regulatory DNA elements throughout the genome, controlling transcription of a large group of specific genes. We introduce an implicit modeling protocol for Myc:Max binding to mesoscale chromatin fibers to determine TF effect on chromatin architecture and shed light on its mechanism of gene regulation. We first bind Myc:Max to different chromatin locations and show how it can direct fiber folding and formation of microdomains, and how this depends on the linker DNA length. Second, by simulating increasing concentrations of Myc:Max binding to fibers that differ in the DNA linker length, linker histone density, and acetylation levels, we assess the interplay between Myc:Max and other chromatin internal parameters. Third, we study the mechanism of gene silencing by Myc:Max binding to the Eed gene loci. Overall, our results show how chromatin architecture can be regulated by TF binding. The position of TF binding dictates the formation of microdomains that appear visible only at the ensemble level. On the other hand, the presence of linker histone, acetylations, or different linker DNA lengths regulates the concentration-dependent effect of TF binding. Furthermore, we show how TF binding can repress gene expression by increasing fiber folding motifs that help compact and occlude the promoter region. Importantly, this effect can be reversed by increasing linker histone density. Overall, these results shed light on the epigenetic control of the genome dictated by TF binding.

Techniques for and challenges in reconstructing 3D genome structures from 2D chromosome conformation capture data.

Chromosome conformation capture technologies that provide frequency information for contacts between genomic regions have been crucial for increasing our understanding of genome folding and regulation. However, such data do not provide direct evidence of the spatial 3D organization of chromatin. In this opinion article, we discuss the development and application of computational methods to reconstruct chromatin 3D structures from experimental 2D contact data, highlighting how such modeling provides biological insights and can suggest mechanisms anchored to experimental data. By applying different reconstruction methods to the same contact data, we illustrate some state-of-the-art of these techniques and discuss our gene resolution approach based on Brownian dynamics and Monte Carlo sampling.

Evolution of coronavirus frameshifting elements: Competing stem networks explain conservation and variability.

The frameshifting RNA element (FSE) in coronaviruses (CoVs) regulates the programmed -1 ribosomal frameshift (-1 PRF) mechanism common to many viruses. The FSE is of particular interest as a promising drug candidate. Its associated pseudoknot or stem loop structure is thought to play a large role in frameshifting and thus viral protein production. To investigate the FSE structural evolution, we use our graph theory-based methods for representing RNA secondary structures in the RNA-As-Graphs (RAG) framework to calculate conformational landscapes of viral FSEs with increasing sequence lengths for representative 10 Alpha and 13 Beta-CoVs. By following length-dependent conformational changes, we show that FSE sequences encode many possible competing stems which in turn favor certain FSE topologies, including a variety of pseudoknots, stem loops, and junctions. We explain alternative competing stems and topological FSE changes by recurring patterns of mutations. At the same time, FSE topology robustness can be understood by shifted stems within different sequence contexts and base pair coevolution. We further propose that the topology changes reflected by length-dependent conformations contribute to tuning the frameshifting efficiency. Our work provides tools to analyze virus sequence/structure correlations, explains how sequence and FSE structure have evolved for CoVs, and provides insights into potential mutations for therapeutic applications against a broad spectrum of CoV FSEs by targeting key sequence/structural transitions.

Effect of Single-Residue Mutations on CTCF Binding to DNA: Insights from Molecular Dynamics Simulations.

In humans and other eukaryotes, DNA is condensed into chromatin fibers that are further wound into chromosomes. This organization allows regulatory elements in the genome, often distant from each other in the linear DNA, to interact and facilitate gene expression through regions known as topologically associating domains (TADs). CCCTC-binding factor (CTCF) is one of the major components of TAD formation and is responsible for recruiting a partner protein, cohesin, to perform loop extrusion and facilitate proper gene expression within TADs. Because single-residue CTCF mutations have been linked to the development of a variety of cancers in humans, we aim to better understand how these mutations affect the CTCF structure and its interaction with DNA. To this end, we compare all-atom molecular dynamics simulations of a wildtype CTCF-DNA complex to those of eight different cancer-linked CTCF mutant sequences. We find that most mutants have lower binding energies compared to the wildtype protein, leading to the formation of less stable complexes. Depending on the type and position of the mutation, this loss of stability can be attributed to major changes in the electrostatic potential, loss of hydrogen bonds between the CTCF and DNA, and/or destabilization of specific zinc fingers. Interestingly, certain mutations in specific fingers can affect the interaction with the DNA of other fingers, explaining why mere single mutations can impair CTCF function. Overall, these results shed mechanistic insights into experimental observations and further underscore CTCF's importance in the regulation of chromatin architecture and gene expression.

Genome modeling: From chromatin fibers to genes.

The intricacies of the 3D hierarchical organization of the genome have been approached by many creative modeling studies. The specific model/simulation technique combination defines and restricts the system and phenomena that can be investigated. We present the latest modeling developments and studies of the genome, involving models ranging from nucleosome systems and small polynucleosome arrays to chromatin fibers in the kb-range, chromosomes, and whole genomes, while emphasizing gene folding from first principles. Clever combinations allow the exploration of many interesting phenomena involved in gene regulation, such as nucleosome structure and dynamics, nucleosome-nucleosome stacking, polynucleosome array folding, protein regulation of chromatin architecture, mechanisms of gene folding, loop formation, compartmentalization, and structural transitions at the chromosome and genome levels. Gene-level modeling with full details on nucleosome positions, epigenetic factors, and protein binding, in particular, can in principle be scaled up to model chromosomes and cells to study fundamental biological regulation.

Brownian dynamics simulations of mesoscale chromatin fibers.

The relationship between chromatin architecture and function defines a central problem in biology and medicine. Many computational chromatin models with atomic, coarse-grained, mesoscale, and polymer resolution have been used to shed light onto the mechanisms that dictate genome folding and regulation of gene expression. The associated simulation techniques range from Monte Carlo to molecular, Brownian, and Langevin dynamics. Here, we present an efficient Compute Unified Device Architecture (CUDA) implementation of Brownian dynamics (BD) to simulate chromatin fibers at the nucleosome resolution with our chromatin mesoscale model. With the CUDA implementation for computer architectures with graphic processing units (GPUs), we significantly accelerate compute-intensive hydrodynamic tensor calculations in the BD simulations by massive parallelization, boosting the performance a hundred-fold compared with central processing unit calculations. We validate our BD simulation approach by reproducing experimental trends on fiber diffusion and structure as a function of salt, linker histone binding, and histone-tail composition, as well as Monte Carlo equilibrium sampling results. Our approach proves to be physically accurate with performance that makes feasible the study of chromatin fibers in the range of kb or hundreds of nucleosomes (small gene). Such simulations are essential to advance the study of biological processes such as gene regulation and aberrant genome-structure related diseases.

Biomotors, viral assembly, and RNA nanobiotechnology: Current achievements and future directions.

The International Society of RNA Nanotechnology and Nanomedicine (ISRNN) serves to further the development of a wide variety of functional nucleic acids and other related nanotechnology platforms. To aid in the dissemination of the most recent advancements, a biennial discussion focused on biomotors, viral assembly, and RNA nanobiotechnology has been established where international experts in interdisciplinary fields such as structural biology, biophysical chemistry, nanotechnology, cell and cancer biology, and pharmacology share their latest accomplishments and future perspectives. The results summarized here highlight advancements in our understanding of viral biology and the structure-function relationship of frame-shifting elements in genomic viral RNA, improvements in the predictions of SHAPE analysis of 3D RNA structures, and the understanding of dynamic RNA structures through a variety of experimental and computational means. Additionally, recent advances in the drug delivery, vaccine design, nanopore technologies, biomotor and biomachine development, DNA packaging, RNA nanotechnology, and drug delivery are included in this critical review. We emphasize some of the novel accomplishments, major discussion topics, and present current challenges and perspectives of these emerging fields.

Chromatin transitions triggered by LH density as epigenetic regulators of the genome.

Motivated by experiments connecting linker histone (LH) deficiency to lymphoma progression and retinal disorders, we study by mesoscale chromatin modeling how LH density (ρ) induces gradual, as well sudden, changes in chromatin architecture and how the process depends on DNA linker length, LH binding dynamics and binding mode, salt concentration, tail modifications, and combinations of ρ and linker DNA length. We show that ρ tightly regulates the overall shape and compaction of the fiber, triggering a transition from an irregular disordered state to a compact and ordered structure. Such a structural transition, resembling B to A compartment transition connected with lymphoma of B cells, appears to occur around ρ = 0.5. The associated mechanism is DNA stem formation by LH binding, which is optimal when the lengths of the DNA linker and LH C-terminal domain are similar. Chromatin internal and external parameters are key regulators, promoting or impeding the transition. The LH density thus emerges as a critical tunable variable in controlling cellular functions through structural transitions of the genome.

RNA-As-Graphs Motif Atlas-Dual Graph Library of RNA Modules and Viral Frameshifting-Element Applications.

RNA motif classification is important for understanding structure/function connections and building phylogenetic relationships. Using our coarse-grained RNA-As-Graphs (RAG) representations, we identify recurrent dual graph motifs in experimentally solved RNA structures based on an improved search algorithm that finds and ranks independent RNA substructures. Our expanded list of 183 existing dual graph motifs reveals five common motifs found in transfer RNA, riboswitch, and ribosomal 5S RNA components. Moreover, we identify three motifs for available viral frameshifting RNA elements, suggesting a correlation between viral structural complexity and frameshifting efficiency. We further partition the RNA substructures into 1844 distinct submotifs, with pseudoknots and junctions retained intact. Common modules are internal loops and three-way junctions, and three submotifs are associated with riboswitches that bind nucleotides, ions, and signaling molecules. Together, our library of existing RNA motifs and submotifs adds to the growing universe of RNA modules, and provides a resource of structures and substructures for novel RNA design.

Length-dependent motions of SARS-CoV-2 frameshifting RNA pseudoknot and alternative conformations suggest avenues for frameshifting suppression.

The SARS-CoV-2 frameshifting element (FSE), a highly conserved mRNA region required for correct translation of viral polyproteins, defines an excellent therapeutic target against Covid-19. As discovered by our prior graph-theory analysis with SHAPE experiments, the FSE adopts a heterogeneous, length-dependent conformational landscape consisting of an assumed 3-stem H-type pseudoknot (graph motif 3_6), and two alternative motifs (3_3 and 3_5). Here, for the first time, we build and simulate, by microsecond molecular dynamics, 30 models for all three motifs plus motif-stabilizing mutants at different lengths. Our 3_6 pseudoknot systems, which agree with experimental structures, reveal interconvertible L and linear conformations likely related to ribosomal pausing and frameshifting. The 3_6 mutant inhibits this transformation and could hamper frameshifting. Our 3_3 systems exhibit length-dependent stem interactions that point to a potential transition pathway connecting the three motifs during ribosomal elongation. Together, our observations provide new insights into frameshifting mechanisms and anti-viral strategies.

Length-dependent motions of SARS-CoV-2 frameshifting RNA pseudoknot and alternative conformations suggest avenues for frameshifting suppression.

Conserved SARS-CoV-2 RNA regions of critical biological functions define excellent targets for anti-viral therapeutics against Covid-19 variants. One such region is the frameshifting element (FSE), responsible for correct translation of viral polyproteins. Here, we analyze molecular-dynamics motions of three FSE conformations, discovered by graph-theory analysis, and associated mutants designed by graph-based inverse folding: two distinct 3-stem H-type pseudoknots and a 3-way junction. We find that the prevalent H-type pseudoknot in literature adopts ring-like conformations, which in combination with 5' end threading could promote ribosomal pausing. An inherent shape switch from "L" to linear that may help trigger the frameshifting is suppressed in our designed mutant. The alternative conformation trajectories suggest a stable intermediate structure with mixed stem interactions of all three conformations, pointing to a possible transition pathway during ribosomal translation. These observations provide new insights into anti-viral strategies and frameshifting mechanisms.

Local chromatin fiber folding represses transcription and loop extrusion in quiescent cells.

A longstanding hypothesis is that chromatin fiber folding mediated by interactions between nearby nucleosomes represses transcription. However, it has been difficult to determine the relationship between local chromatin fiber compaction and transcription in cells. Further, global changes in fiber diameters have not been observed, even between interphase and mitotic chromosomes. We show that an increase in the range of local inter-nucleosomal contacts in quiescent yeast drives the compaction of chromatin fibers genome-wide. Unlike actively dividing cells, inter-nucleosomal interactions in quiescent cells require a basic patch in the histone H4 tail. This quiescence-specific fiber folding globally represses transcription and inhibits chromatin loop extrusion by condensin. These results reveal that global changes in chromatin fiber compaction can occur during cell state transitions, and establish physiological roles for local chromatin fiber folding in regulating transcription and chromatin domain formation.

Biomolecular modeling thrives in the age of technology.

The biomolecular modeling field has flourished since its early days in the 1970s due to the rapid adaptation and tailoring of state-of-the-art technology. The resulting dramatic increase in size and timespan of biomolecular simulations has outpaced Moore's law. Here, we discuss the role of knowledge-based versus physics-based methods and hardware versus software advances in propelling the field forward. This rapid adaptation and outreach suggests a bright future for modeling, where theory, experimentation and simulation define three pillars needed to address future scientific and biomedical challenges.

To Knot or Not to Knot: Multiple Conformations of the SARS-CoV-2 Frameshifting RNA Element.

The SARS-CoV-2 frameshifting RNA element (FSE) is an excellent target for therapeutic intervention against Covid-19. This small gene element employs a shifting mechanism to pause and backtrack the ribosome during translation between Open Reading Frames 1a and 1b, which code for viral polyproteins. Any interference with this process has a profound effect on viral replication and propagation. Pinpointing the structures adapted by the FSE and associated structural transformations involved in frameshifting has been a challenge. Using our graph-theory-based modeling tools for representing RNA secondary structures, "RAG" (RNA-As-Graphs), and chemical structure probing experiments, we show that the 3-stem H-type pseudoknot (3_6 dual graph), long assumed to be the dominant structure, has a viable alternative, an HL-type 3-stem pseudoknot (3_3) for longer constructs. In addition, an unknotted 3-way junction RNA (3_5) emerges as a minor conformation. These three conformations share Stems 1 and 3, while the different Stem 2 may be involved in a conformational switch and possibly associations with the ribosome during translation. For full-length genomes, a stem-loop motif (2_2) may compete with these forms. These structural and mechanistic insights advance our understanding of the SARS-CoV-2 frameshifting process and concomitant virus life cycle, and point to three avenues of therapeutic intervention.