Co-transcriptional gene regulation in eukaryotes and prokaryotes.

Many steps of RNA processing occur during transcription by RNA polymerases. Co-transcriptional activities are deemed commonplace in prokaryotes, in which the lack of membrane barriers allows mixing of all gene expression steps, from transcription to translation. In the past decade, an extraordinary level of coordination between transcription and RNA processing has emerged in eukaryotes. In this Review, we discuss recent developments in our understanding of co-transcriptional gene regulation in both eukaryotes and prokaryotes, comparing methodologies and mechanisms, and highlight striking parallels in how RNA polymerases interact with the machineries that act on nascent RNA. The development of RNA sequencing and imaging techniques that detect transient transcription and RNA processing intermediates has facilitated discoveries of transcription coordination with splicing, 3'-end cleavage and dynamic RNA folding and revealed physical contacts between processing machineries and RNA polymerases. Such studies indicate that intron retention in a given nascent transcript can prevent 3'-end cleavage and cause transcriptional readthrough, which is a hallmark of eukaryotic cellular stress responses. We also discuss how coordination between nascent RNA biogenesis and transcription drives fundamental aspects of gene expression in both prokaryotes and eukaryotes.

Five questions on how biochemistry can combat climate change.

Global warming is caused by human activity, such as the burning of fossil fuels, which produces high levels of greenhouse gasses. As a consequence, climate change impacts all organisms and the greater ecosystem through changing conditions from weather patterns to the temperature, pH and salt concentrations found in waterways and soil. These environmental changes fundamentally alter many parameters of the living world, from the kinetics of chemical reactions and cellular signaling pathways to the accumulation of unforeseen chemicals in the environment, the appearance and dispersal of new diseases, and the availability of traditional foods. Some organisms adapt to extremes, while others cannot. This article asks five questions that prompt us to consider the foundational knowledge that biochemistry can bring to the table as we meet the challenge of climate change. We approach climate change from the molecular point of view, identifying how cells and organisms - from microbes to plants and animals - respond to changing environmental conditions. To embrace the concept of "one health" for all life on the planet, we argue that we must leverage biochemistry, cell biology, molecular biophysics and genetics to fully understand the impact of climate change on the living world and to bring positive change.

Delta SARS-CoV-2 s2m Structure, Dynamics, and Entropy: Consequences of the G15U Mutation.

Bioinformatic analysis of the Delta SARS-CoV-2 genome reveals a single nucleotide mutation (G15U) in the stem-loop II motif (s2m) relative to ancestral SARS-CoV-2. Despite sequence similarity, unexpected differences between SARS-CoV-2 and Delta SARS-CoV-2 s2m homodimerization experiments require the discovery of unknown structural and thermodynamic changes necessary to rationalize the data. Using our reported SARS-CoV-2 s2m model, we induced the G15U substitution and performed 3.5 microseconds of unbiased molecular dynamics simulation at 283 and 310 K. The resultant Delta s2m adopted a secondary structure consistent with our reported NMR data, resulting in significant deviations in the tertiary structure and dynamics from our SARS-CoV-2 s2m model. First, we find differences in the overall three-dimensional structure, where the characteristic 90° L-shaped kink of the SARS-CoV-2 s2m did not form in the Delta s2m resulting in a "linear" hairpin with limited bending dynamics. Delta s2m helical parameters are calculated to align closely with A-form RNA, effectively eliminating a hinge point to form the L-shape kink by correcting an upper stem defect in SARS-CoV-2 induced by a noncanonical and dynamic G:A base pair. Ultimately, the shape difference rationalizes the migration differences in reported electrophoresis experiments. Second, increased fluctuation of the Delta s2m palindromic sequence, within the terminal loop, compared to SARS-CoV-2 s2m results in an estimated increase of entropy of 6.8 kcal/mol at 310 K relative to the SARS-CoV-2 s2m. The entropic difference offers a unique perspective on why the Delta s2m homodimerizes less spontaneously, forming fewer kissing dimers and extended duplexes compared to SARS-CoV-2. In this work, both the L-shape reduction and palindromic entropic penalty provides an explanation of our reported in vitro electrophoresis homodimerization results. Ultimately, the structural, dynamical, and entropic differences between the SARS-CoV-2 s2m and Delta s2m serve to establish a foundation for future studies of the s2m function in the viral lifecycle.

Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p.

The s2m, a highly conserved 41-nt hairpin structure in the SARS-CoV-2 genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and the subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a >99% correlation of a single nucleotide mutation at the 15th position (G15U) in Delta SARS-CoV-2. Based on 1H NMR spectroscopy assignments comparing the imino proton resonance region of s2m and the s2m G15U at 19°C, we show that the U15-A29 base pair closes, resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we show that the s2m G15U mutation drastically impacts the binding of host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on immune responses.

Uridylation of the histone mRNA stem-loop weakens binding interactions with SLBP while maintaining interactions with 3'hExo.

Histone mRNA degradation is controlled by the unique 3' stem-loop of histone mRNA and the stem-loop binding protein (SLBP). As part of this process, the 3' stem-loop is trimmed by the histone-specific 3' exonuclease (3'hExo) and uridylated by the terminal uridylyl transferase 7 (TUT7), creating partially degraded intermediates with short uridylations. The role of these uridylations in degradation is not fully understood. Our work examines changes in the stability of the ternary complex created by trimming and uridylation of the stem-loop to better understand the role of this process in the histone mRNA life cycle. In this study, we used fluorescence polarization and electrophoretic mobility shift assays to demonstrate that both SLBP and 3'hExo can bind to uridylated and partially degraded stem-loop intermediates, although with lower affinity. We further characterized this complex by performing 1-µs molecular dynamics simulations using the AMBER force field and Nanoscale Molecular Dynamics (NAMD). These simulations show that while uridylation helps maintain the overall shape of the stem-loop, the combination of uridylation and dephosphorylation of the TPNK motif in SLBP disrupts key RNA-protein interactions. They also demonstrate that uridylation allows 3'hExo to maintain contact with the stem-loop after partial degradation and plays a role in disrupting key base pairs in partially degraded histone mRNA intermediates. Together, these experiments and simulations suggest that trimming by 3'hExo, uridylation, and SLBP dephosphorylation weakens both RNA-protein interactions and the stem-loop itself. Our results further elucidate the role of uridylation and SLBP dephosphorylation in the early stages of histone mRNA degradation.

Effect of the SARS-CoV-2 Delta-associated G15U mutation on the s2m element dimerization and its interactions with miR-1307-3p.

The stem loop 2 motif (s2m), a highly conserved 41-nucleotide hairpin structure in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome, serves as an attractive therapeutic target that may have important roles in the virus life cycle or interactions with the host. However, the conserved s2m in Delta SARS-CoV-2, a previously dominant variant characterized by high infectivity and disease severity, has received relatively less attention than that of the original SARS-CoV-2 virus. The focus of this work is to identify and define the s2m changes between Delta and SARS-CoV-2 and subsequent impact of those changes upon the s2m dimerization and interactions with the host microRNA miR-1307-3p. Bioinformatics analysis of the GISAID database targeting the s2m element reveals a greater than 99% correlation of a single nucleotide mutation at the 15 th position (G15U) in Delta SARS-CoV-2. Based on 1 H NMR assignments comparing the imino proton resonance region of s2m and the G15U at 19°C, we find that the U15-A29 base pair closes resulting in a stabilization of the upper stem without overall secondary structure deviation. Increased stability of the upper stem did not affect the chaperone activity of the viral N protein, as it was still able to convert the kissing dimers formed by s2m G15U into a stable duplex conformation, consistent with the s2m reference. However, we find that the s2m G15U mutation drastically reduces the binding affinity of the host miR-1307-3p. These findings demonstrate that the observed G15U mutation alters the secondary structure of s2m with subsequent impact on viral binding of host miR-1307-3p, with potential consequences on the immune response.

US-align: universal structure alignments of proteins, nucleic acids, and macromolecular complexes.

Structure comparison and alignment are of fundamental importance in structural biology studies. We developed the first universal platform, US-align, to uniformly align monomer and complex structures of different macromolecules-proteins, RNAs and DNAs. The pipeline is built on a uniform TM-score objective function coupled with a heuristic alignment searching algorithm. Large-scale benchmarks demonstrated consistent advantages of US-align over state-of-the-art methods in pairwise and multiple structure alignments of different molecules. Detailed analyses showed that the main advantage of US-align lies in the extensive optimization of the unified objective function powered by efficient heuristic search iterations, which substantially improve the accuracy and speed of the structural alignment process. Meanwhile, the universal protocol fusing different molecular and structural types helps facilitate the heterogeneous oligomer structure comparison and template-based protein-protein and protein-RNA/DNA docking.

AMIGOS III: pseudo-torsion angle visualization and motif-based structure comparison of nucleic acids.

MOTIVATION: The full description of nucleic acid conformation involves eight torsion angles per nucleotide. To simplify this description, we previously developed a representation of the nucleic acid backbone that assigns each nucleotide a pair of pseudo-torsion angles (eta and theta defined by P and C4' atoms; or eta' and theta' defined by P and C1' atoms). A Java program, AMIGOS II, is currently available for calculating eta and theta angles for RNA and for performing motif searches based on eta and theta angles. However, AMIGOS II lacks the ability to parse DNA structures and to calculate eta' and theta' angles. It also has little visualization capacity for 3D structure, making it difficult for users to interpret the computational results. RESULTS: We present AMIGOS III, a PyMOL plugin that calculates the pseudo-torsion angles eta, theta, eta' and theta' for both DNA and RNA structures and performs motif searching based on these angles. Compared to AMIGOS II, AMIGOS III offers improved pseudo-torsion angle visualization for RNA and faster nucleic acid worm database generation; it also introduces pseudo-torsion angle visualization for DNA and nucleic acid worm visualization. Its integration into PyMOL enables easy preparation of tertiary structure inputs and intuitive visualization of involved structures. AVAILABILITY AND IMPLEMENTATION: https://github.com/pylelab/AMIGOSIII. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.