Determining structures of RNA conformers using AFM and deep neural networks.

Much of the human genome is transcribed into RNAs, many of which contain structural elements that are important for their function. Such RNA molecules-including those that are structured and well-folded-are conformationally heterogeneous and flexible, which is a prerequisite for function, but this limits the applicability of methods such as NMR, crystallography and cryo-electron microscopy for structure elucidation. Moreover, owing to the lack of a large RNA structure database, and no clear correlation between sequence and structure, approaches such as AlphaFold for protein structure prediction do not apply to RNA.

The conformational space of RNase P RNA in solution.

RNA conformational diversity has fundamental biological roles, but direct visualization of its full conformational space in solution has not been possible using traditional biophysical techniques. Using solution atomic force microscopy, a deep neural network and statistical analyses, we show that the ribonuclease P (RNase P) RNA adopts heterogeneous conformations consisting of a conformationally invariant core and highly flexible peripheral structural elements that sample a broad conformational space, with amplitudes as large as 20-60 Å in a multitude of directions, with very low net energy cost. Increasing Mg drives compaction and enhances enzymatic activity, probably by narrowing the conformational space.

Riboswitch Mechanisms for Regulation of P1 Helix Stability.

Riboswitches are highly structured RNA regulators of gene expression. Although found in all three domains of life, they are particularly abundant and widespread in bacteria, including many human pathogens, thus making them an attractive target for antimicrobial development. Moreover, the functional versatility of riboswitches to recognize a myriad of ligands, including ions, amino acids, and diverse small-molecule metabolites, has enabled the generation of synthetic aptamers that have been used as molecular probes, sensors, and regulatory RNA devices.

Autoinhibition of ubiquitin-specific protease 8: Insights into domain interactions and mechanisms of regulation.

Ubiquitin-specific proteases (USPs) are a family of multi-domain deubiquitinases (DUBs) with variable architectures, some containing regulatory auxiliary domains. Among the USP family, all occurrences of intramolecular regulation presently known are autoactivating. USP8 remains the sole exception as its putative WW-like domain, conserved only in vertebrate orthologs, is autoinhibitory.

Overexpression of a BR inactivating enzyme gene GhPAG1 impacts eggplant fruit development and anthocyanin accumulation mainly by altering hormone homeostasis.

Brassinosteroids (BRs) function importantly in plant growth and development, but the roles in regulating fruit development and anthocyanin pigmentation remain unclear. Eggplant (Solanum melongena L.) is an important Solanaceae vegetable crop rich in anthocyanins.

Direct observation of tRNA-chaperoned folding of a dynamic mRNA ensemble.

T-box riboswitches are multi-domain noncoding RNAs that surveil individual amino acid availabilities in most Gram-positive bacteria. T-boxes directly bind specific tRNAs, query their aminoacylation status to detect starvation, and feedback control the transcription or translation of downstream amino-acid metabolic genes. Most T-boxes rapidly recruit their cognate tRNA ligands through an intricate three-way stem I-stem II-tRNA interaction, whose establishment is not understood.

Capturing heterogeneous conformers of cobalamin riboswitch by cryo-EM.

RNA conformational heterogeneity often hampers its high-resolution structure determination, especially for large and flexible RNAs devoid of stabilizing proteins or ligands. The adenosylcobalamin riboswitch exhibits heterogeneous conformations under 1 mM Mg2+ concentration and ligand binding reduces conformational flexibility. Among all conformers, we determined one apo (5.

Architectural basis for cylindrical self-assembly governing Plk4-mediated centriole duplication in human cells.

Proper organization of intracellular assemblies is fundamental for efficient promotion of biochemical processes and optimal assembly functionality. Although advances in imaging technologies have shed light on how the centrosome is organized, how its constituent proteins are coherently architected to elicit downstream events remains poorly understood. Using multidisciplinary approaches, we showed that two long coiled-coil proteins, Cep63 and Cep152, form a heterotetrameric building block that undergoes a stepwise formation into higher molecular weight complexes, ultimately generating a cylindrical architecture around a centriole.

Prognostic Analysis and Biomarkers Identification of Immune Infiltration in Early and Late Stage Hepatocellular Carcinoma Based on TCGA Data.

Background: Hepatocellular carcinoma (HCC) is a major cause of cancer death in the world. The aim of this study was to establish a new model to predict the prognosis of HCC.

Materials And Methods: The mRNA, miRNA and lncRNA expression profiles of early (stage I-II) and late (stage III-IV) stage HCC patients were acquired from The Cancer Genome Atlas (TCGA) database.

Crystal structure of Escherichia coli thiamine pyrophosphate-sensing riboswitch in the apo state.

The thiamine pyrophosphate (TPP)-sensing riboswitch is one of the earliest discovered and most widespread riboswitches. Numerous structural studies have been reported for this riboswitch bound with various ligands. However, the ligand-free (apo) structure remains unknown.

SmCIP7, a COP1 interactive protein, positively regulates anthocyanin accumulation and fruit size in eggplant.

In higher plants, COP1 (Constitutively Photomorphogenic 1) acts as a central regulator of light-signaling networks and globally conditions the target proteins via the ubiquitin-proteasome pathway. However, the function of COP1-interacting proteins in light-regulated fruit coloration and development remains unknown in Solanaceous plants. Here, a COP1-interacting protein-encoding gene, SmCIP7, expressed specifically in the eggplant (Solanum melongena L.

Deriving RNA topological structure from SAXS.

Structures of well-folded RNA molecules can be determined with atomic resolution by either X-ray crystallography, cryo-EM, or NMR spectroscopy, but those of conformationally-flexible RNAs often are difficult to study with these methods. However, flexible RNAs have biological relevance and likely represent the majority of the RNA conformational space. Due to the high electron density of the phosphate-sugar backbone, RNA is very sensitive to small-angle X-ray scattering (SAXS), and SAXS data can be recorded with sub-μM concentrations and under near-physiological solution conditions without the need for labeling.

Mix-and-Inject Serial Femtosecond Crystallography to Capture RNA Riboswitch Intermediates.

Time-resolved structure determination of macromolecular conformations and ligand-bound intermediates is extremely challenging, particularly for RNA. With rapid technological advances in both microfluidic liquid injection and X-ray free electron lasers (XFEL), a new frontier has emerged in time-resolved crystallography whereby crystals can be mixed with ligand and then probed with X-rays (mix-and-inject) in real time and at room temperature. This chapter outlines the basic setup and procedures for mix-and-inject experiments for recording time-resolved crystallographic data of riboswitch RNA reaction states using serial femtosecond crystallography (SFX) and an XFEL.

Combining Biophysical Methods for Structure-Function Analyses of RNA in Solution.

RNA-level regulation by riboswitches relies on the specific binding of small metabolites to the aptamer domain to trigger substantial conformational changes that affect transcription or translation. Although several biophysical methods have been employed to study such RNAs, the utility of any one single method is limited. Hybrid approaches, therefore, are essential to better characterize these intrinsically dynamic molecules and elucidate their regulatory mechanisms driven by ligand-induced conformational changes.

Developing methods to study conformational changes in RNA crystals using a photocaged ligand.

Crystallographic observation of structural changes in real time requires that those changes be uniform both spatially and temporally. A primary challenge with time-resolved ligand-mixing diffraction experiments is asynchrony caused by variable factors, such as efficiency of mixing, rate of diffusion, crystal size, and subsequently, conformational heterogeneity. One method of minimizing such variability is use of a photolabile caged ligand, which can fully saturate the crystal environment (spatially), and whose photoactivation can rapidly (temporally) trigger the reaction in a controlled manner.

CYP26A1 Is a Novel Cancer Biomarker of Pancreatic Carcinoma: Evidence from Integration Analysis and Experiments.

Background: CYP26A1 has been reported in multiple cancers. However, the role of CYP26A1 in pancreatic cancer (PC) has not been explored.

Method: The public data used for this study was obtained from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Cancer Cell Line Encyclopedia (CCLE) cell lines.

Taf2 mediates DNA binding of Taf14.

The assembly and function of the yeast general transcription factor TFIID complex requires specific contacts between its Taf14 and Taf2 subunits, however, the mechanism underlying these contacts remains unclear. Here, we determined the molecular and structural basis by which the YEATS and ET domains of Taf14 bind to the C-terminal tail of Taf2 and identified a unique DNA-binding activity of the linker region connecting the two domains. We show that in the absence of ligands the linker region of Taf14 is occluded by the surrounding domains, and therefore the DNA binding function of Taf14 is autoinhibited.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66 .

The low-resolution structural models of hepatitis C virus RNA subdomain 5BSL3.2 and its distal complex with domain 3'X point to conserved regulatory mechanisms within the Flaviviridae family.

Subdomain 5BSL3.2 of hepatitis C virus RNA lies at the core of a network of distal RNA-RNA contacts that connect the 5' and 3' regions of the viral genome and regulate the translation and replication stages of the viral cycle. Using small-angle X-ray scattering and NMR spectroscopy experiments, we have determined at low resolution the structural models of this subdomain and its distal complex with domain 3'X, located at the 3'-terminus of the viral RNA chain.

The mechanism driving a solid-solid phase transition in a biomacromolecular crystal.

Solid-solid phase transitions (SSPTs) occur between distinguishable crystalline forms. Because of their importance in application and theory in materials science and condensed-matter physics, SSPTs have been studied most extensively in metallic alloys, inorganic salts and small organic molecular crystals, but much less so in biomacromolecular crystals. In general, the mechanisms of SSPTs at the atomic and molecular levels are not well understood.

Dependence of phase transition uniformity on crystal sizes characterized using birefringence.

Solid-solid phase transitions (SSPTs) have been widely observed in crystals of organic or inorganic small-molecules. Although SSPTs in macromolecular crystals have been reported, the majority involve local atomic changes, such as those induced by changes in hydration. SSPTs driven by large conformational changes, however, can be more difficult to characterize since they often significantly disrupt lattice packing interactions.