LncRNA FLAIL affects alternative splicing and represses flowering in Arabidopsis.

How the noncoding genome affects cellular functions is a key biological question. A particular challenge is to distinguish the effects of noncoding DNA elements from long noncoding RNAs (lncRNAs) that coincide at the same loci. Here, we identified the flowering-associated intergenic lncRNA (FLAIL) in Arabidopsis through early flowering flail mutants. Expression of FLAIL RNA from a different chromosomal location in combination with strand-specific RNA knockdown characterized FLAIL as a trans-acting RNA molecule. FLAIL directly binds to differentially expressed target genes that control flowering via RNA-DNA interactions through conserved sequence motifs. FLAIL interacts with protein and RNA components of the spliceosome to affect target mRNA expression through co-transcriptional alternative splicing (AS) and linked chromatin regulation. In the absence of FLAIL, splicing defects at the direct FLAIL target flowering gene LACCASE 8 (LAC8) correlated with reduced mRNA expression. Double mutant analyses support a model where FLAIL-mediated splicing of LAC8 promotes its mRNA expression and represses flowering. Our study suggests lncRNAs as accessory components of the spliceosome that regulate AS and gene expression to impact organismal development.

A data-driven genome annotation approach for cassava.

Genome annotation files play a critical role in dictating the quality of downstream analyses by providing essential predictions for gene positions and structures. These files are pivotal in decoding the complex information encoded within DNA sequences. Here, we generated experimental data resolving RNA 5'- and 3'-ends as well as full-length RNAs for cassava TME12 sticklings in ambient temperature and cold. We used these data to generate genome annotation files using the TranscriptomeReconstructoR (TR) tool. A careful comparison to high-quality genome annotations suggests that our new TR genome annotations identified additional genes, resolved the transcript boundaries more accurately and identified additional RNA isoforms. We enhanced existing cassava genome annotation files with the information from TR that maintained the different transcript models as RNA isoforms. The resultant merged annotation was subsequently utilized for comprehensive analysis. To examine the effects of genome annotation files on gene expression studies, we compared the detection of differentially expressed genes during cold using the same RNA-seq data but alternative genome annotation files. We found that our merged genome annotation that included cold-specific TR gene models identified about twice as many cold-induced genes. These data indicate that environmentally induced genes may be missing in off-the-shelf genome annotation files. In conclusion, TR offers the opportunity to enhance crop genome annotations with implications for the discovery of differentially expressed candidate genes during plant-environment interactions.

The Hda1 histone deacetylase limits divergent non-coding transcription and restricts transcription initiation frequency.

Nucleosome-depleted regions (NDRs) at gene promoters support initiation of RNA polymerase II transcription. Interestingly, transcription often initiates in both directions, resulting in an mRNA and a divergent non-coding (DNC) transcript of unclear purpose. Here, we characterized the genetic architecture and molecular mechanism of DNC transcription in budding yeast. Using high-throughput reverse genetic screens based on quantitative single-cell fluorescence measurements, we identified the Hda1 histone deacetylase complex (Hda1C) as a repressor of DNC transcription. Nascent transcription profiling showed a genome-wide role of Hda1C in repression of DNC transcription. Live-cell imaging of transcription revealed that mutations in the Hda3 subunit increased the frequency of DNC transcription. Hda1C contributed to decreased acetylation of histone H3 in DNC transcription regions, supporting DNC transcription repression by histone deacetylation. Our data support the interpretation that DNC transcription results as a consequence of the NDR-based architecture of eukaryotic promoters, but that it is governed by locus-specific repression to maintain genome fidelity.

Characterization of Arabidopsis thaliana Promoter Bidirectionality and Antisense RNAs by Inactivation of Nuclear RNA Decay Pathways.

In animals, RNA polymerase II initiates transcription bidirectionally from gene promoters to produce pre-mRNAs on the forward strand and promoter upstream transcripts (PROMPTs) on the reverse strand. PROMPTs are degraded by the nuclear exosome. Previous studies based on nascent RNA approaches concluded that Arabidopsis (Arabidopsis thaliana) does not produce PROMPTs. Here, we used steady-state RNA sequencing in mutants defective in nuclear RNA decay including the exosome to reassess the existence of Arabidopsis PROMPTs. While they are rare, we identified ∼100 cases of exosome-sensitive PROMPTs in Arabidopsis. Such PROMPTs are sources of small interfering RNAs in exosome-deficient mutants, perhaps explaining why plants have evolved mechanisms to suppress PROMPTs. In addition, we found ∼200 long, unspliced and exosome-sensitive antisense RNAs that arise from transcription start sites within parts of the genome encoding 3'-untranslated regions on the sense strand. The previously characterized noncoding RNA that regulates expression of the key seed dormancy regulator, DELAY OF GERMINATION1, is a typical representative of this class of RNAs. Transcription factor genes are overrepresented among loci with exosome-sensitive antisense RNAs, suggesting a potential for widespread control of gene expression via this class of noncoding RNAs. Lastly, we assess the use of alternative promoters in Arabidopsis and compare the accuracy of existing TSS annotations.

Excitonic Coupling in Fluorene-Based Bichromophoric Systems: Vibrational Quenching and the Transition from Weak to Intermediate Coupling.

Excimeric systems (i.e., excited dimers) have well served as model compounds for the study of the delocalization of electronic energy over weakly interacting chromophores. However, there remain relatively few isolated systems in which such interactions can be studied experimentally at a level to afford detailed comparisons with theory. In this Article, we examine a series of covalently and noncovalently linked dimers of fluorene, as a model aromatic chromophore, where the formation of excimers requires a π-stacked, cofacial orientation at van der Waals contact. Building upon a series of seminal prior studies that examined vibronic quenching of the excitation interaction in van der Waals dimers, the key question that we sought to address here is whether a single quenching factor could reproduce experimental excitonic splittings across a series of covalently and noncovalently linked bichromophoric systems built from the same chromophore. In comparing experimentally measured excitonic splittings with calculated static splittings using time-dependent density functional methods, we find that all systems save one fall on a line with a slope of 0.080(8), reflecting a vibrational quenching of roughly 1 order of magnitude. The outlier, which shows a significantly reduced quenching factor, represents a cyclophane-linked system where the fluorene moieties are constrained in a cofacial arrangement. We argue that this system evidences the transition from the weak to intermediate coupling regime.

Incidental germline findings during molecular profiling of tumor tissues for precision oncology: molecular survey and methodological obstacles.

BACKGROUND: A fraction of patients referred for complex molecular profiling of biopsied tumors may harbor germline variants in genes associated with the development of hereditary cancer syndromes (HCS). Neither the bioinformatic analysis nor the reporting of such incidental germline findings are standardized. METHODS: Data from Next-Generation Sequencing (NGS) of biopsied tumor samples referred for complex molecular profiling were analyzed for germline variants in HCS-associated genes. Analysis of variant origin was performed employing bioinformatic algorithms followed by manual curation. When possible, the origin of the variant was validated by Sanger sequencing of the sample of normal tissue. The variants' pathogenicity was assessed according to ACMG/AMP. RESULTS: Tumors were sampled from 183 patients (Males: 75 [41.0%]; Females: 108 [59.0%]; mean [SD] age, 57.7 [13.3] years) and analysed by targeted NGS. The most common tumor types were colorectal (19%), pancreatic (13%), and lung cancer (10%). A total of 56 sequence variants in genes associated with HCS were detected in 40 patients. Of them, 17 variants found in 14 patients were predicted to be of germline origin, with 6 variants interpreted as pathogenic (PV) or likely pathogenic (LPV), and 9 as variants of uncertain significance (VUS). For the 41 out of 42 (97%) missense variants in HCS-associated genes, the results of computational prediction of variant origin were concordant with that of experimental examination. We estimate that Sanger sequencing of a sample of normal tissue would be required for ~ 1-7% of the total assessed cases with PV or LPV, when necessity to follow with genetic counselling referral in ~ 2-15% of total assessed cases (PV, LPV or VUS found in HCS genes). CONCLUSION: Incidental findings of pathogenic germline variants are common in data from cancer patients referred for complex molecular profiling. We propose an algorithm for the management of patients with newly detected variants in genes associated with HCS.

Organismal benefits of transcription speed control at gene boundaries.

RNA polymerase II (RNAPII) transcription is crucial for gene expression. RNAPII density peaks at gene boundaries, associating these key regions for gene expression control with limited RNAPII movement. The connections between RNAPII transcription speed and gene regulation in multicellular organisms are poorly understood. Here, we directly modulate RNAPII transcription speed by point mutations in the second largest subunit of RNAPII in Arabidopsis thaliana. A RNAPII mutation predicted to decelerate transcription is inviable, while accelerating RNAPII transcription confers phenotypes resembling auto-immunity. Nascent transcription profiling revealed that RNAPII complexes with accelerated transcription clear stalling sites at both gene ends, resulting in read-through transcription. The accelerated transcription mutant NRPB2-Y732F exhibits increased association with 5' splice site (5'SS) intermediates and enhanced splicing efficiency. Our findings highlight potential advantages of RNAPII stalling through local reduction in transcription speed to optimize gene expression for the development of multicellular organisms.

Reconstruction of time changes in radiocesium concentrations in the river of the Fukushima Dai-ichi NPP contaminated area based on its depth distribution in dam reservoir's bottom sediments.

Radionuclide depth distribution in bottom sediments in deep-water zones of dam reservoirs, where no sediment mixing occurs, can be used to reconstruct time changes in particulate activity concentrations of radionuclides strongly bound to bottom sediments. This approach was used to analyze the 137Cs concentration profile in a bottom sediment core collected from Ogaki dam reservoir on the Ukedo River in the Fukushima Dai-ichi nuclear power plant contaminated zone in October 2019. The derived 137Cs particulate concentrations provided a basis for estimating the dissolved concentration and its temporal trend in the Ukedo River, using the mean value of the apparent 137Cs distribution coefficient. The reconstructed particulate and dissolved 137Cs concentrations and their temporal trends are consistent with monitoring data. The annual mean particulate and dissolved 137Cs wash-off ratios were also calculated for the period of eight years after the accident. Interestingly, the particulate 137Cs wash-off ratios for the Ukedo River at Ogaki dam were found to be similar to those for the Pripyat River at Chernobyl in the same time period after the accident, while the dissolved 137Cs wash-off ratios in the Ukedo River were an order of magnitude lower than the corresponding values in the Pripyat River. Both the particulate and dissolved 137Cs wash-off ratios in the Ukedo River declined faster during the first eight years after the FDNPP accident than predicted by the diffusional model, most likely, due to greater natural attenuation and, to some extent, remediation measures implemented on the catchments in Fukushima.

Native elongation transcript sequencing reveals temperature dependent dynamics of nascent RNAPII transcription in Arabidopsis.

Temperature profoundly affects the kinetics of biochemical reactions, yet how large molecular complexes such as the transcription machinery accommodate changing temperatures to maintain cellular function is poorly understood. Here, we developed plant native elongating transcripts sequencing (plaNET-seq) to profile genome-wide nascent RNA polymerase II (RNAPII) transcription during the cold-response of Arabidopsis thaliana with single-nucleotide resolution. Combined with temporal resolution, these data revealed transient genome-wide reprogramming of nascent RNAPII transcription during cold, including characteristics of RNAPII elongation and thousands of non-coding transcripts connected to gene expression. Our results suggest a role for promoter-proximal RNAPII stalling in predisposing genes for transcriptional activation during plant-environment interactions. At gene 3'-ends, cold initially facilitated transcriptional termination by limiting the distance of read-through transcription. Within gene bodies, cold reduced the kinetics of co-transcriptional splicing leading to increased intragenic stalling. Our data resolved multiple distinct mechanisms by which temperature transiently altered the dynamics of nascent RNAPII transcription and associated RNA processing, illustrating potential biotechnological solutions and future focus areas to promote food security in the context of a changing climate.

Transcription-driven chromatin repression of Intragenic transcription start sites.

Progression of RNA polymerase II (RNAPII) transcription relies on the appropriately positioned activities of elongation factors. The resulting profile of factors and chromatin signatures along transcription units provides a "positional information system" for transcribing RNAPII. Here, we investigate a chromatin-based mechanism that suppresses intragenic initiation of RNAPII transcription. We demonstrate that RNAPII transcription across gene promoters represses their function in plants. This repression is characterized by reduced promoter-specific molecular signatures and increased molecular signatures associated with RNAPII elongation. The conserved FACT histone chaperone complex is required for this repression mechanism. Genome-wide Transcription Start Site (TSS) mapping reveals thousands of discrete intragenic TSS positions in fact mutants, including downstream promoters that initiate alternative transcript isoforms. We find that histone H3 lysine 4 mono-methylation (H3K4me1), an Arabidopsis RNAPII elongation signature, is enriched at FACT-repressed intragenic TSSs. Our analyses suggest that FACT is required to repress intragenic TSSs at positions that are in part characterized by elevated H3K4me1 levels. In sum, conserved and plant-specific chromatin features correlate with the co-transcriptional repression of intragenic TSSs. Our insights into TSS repression by RNAPII transcription promise to inform the regulation of alternative transcript isoforms and the characterization of gene regulation through the act of pervasive transcription across eukaryotic genomes.

TrancriptomeReconstructoR: data-driven annotation of complex transcriptomes.

BACKGROUND: The quality of gene annotation determines the interpretation of results obtained in transcriptomic studies. The growing number of genome sequence information calls for experimental and computational pipelines for de novo transcriptome annotation. Ideally, gene and transcript models should be called from a limited set of key experimental data. RESULTS: We developed TranscriptomeReconstructoR, an R package which implements a pipeline for automated transcriptome annotation. It relies on integrating features from independent and complementary datasets: (i) full-length RNA-seq for detection of splicing patterns and (ii) high-throughput 5' and 3' tag sequencing data for accurate definition of gene borders. The pipeline can also take a nascent RNA-seq dataset to supplement the called gene model with transient transcripts. We reconstructed de novo the transcriptional landscape of wild type Arabidopsis thaliana seedlings and Saccharomyces cerevisiae cells as a proof-of-principle. A comparison to the existing transcriptome annotations revealed that our gene model is more accurate and comprehensive than the most commonly used community gene models, TAIR10 and Araport11 for A.thaliana and SacCer3 for S.cerevisiae. In particular, we identify multiple transient transcripts missing from the existing annotations. Our new annotations promise to improve the quality of A.thaliana and S.cerevisiae genome research. CONCLUSIONS: Our proof-of-concept data suggest a cost-efficient strategy for rapid and accurate annotation of complex eukaryotic transcriptomes. We combine the choice of library preparation methods and sequencing platforms with the dedicated computational pipeline implemented in the TranscriptomeReconstructoR package. The pipeline only requires prior knowledge on the reference genomic DNA sequence, but not the transcriptome. The package seamlessly integrates with Bioconductor packages for downstream analysis.

Mechanism-Based Inactivation of Cytochrome P450 Enzymes: Computational Insights.

Mechanism-based inactivation (MBI) refers to the metabolic bioactivation of a xenobiotic by cytochrome P450s to a highly reactive intermediate which subsequently binds to the enzyme and leads to the quasi-irreversible or irreversible inhibition. Xenobiotics, mainly drugs with specific functional units, are the major sources of MBI. Two possible consequences of MBI by medicinal compounds are drug-drug interaction and severe toxicity that are observed and highlighted by clinical experiments. Today almost all of these latent functional groups (e.g., thiophene, furan, alkylamines, etc.) are known, and their features and mechanisms of action, owing to the vast experimental and theoretical studies, are determined. In the past decade, molecular modeling techniques, mostly density functional theory, have revealed the most feasible mechanism that a drug undergoes by P450 enzymes to generate a highly reactive intermediate. In this review, we provide a comprehensive and detailed picture of computational advances toward the elucidation of the activation mechanisms of various known groups with MBI activity. To this aim, we briefly describe the computational concepts to carry out and analyze the mechanistic investigations, and then, we summarize the studies on compounds with known inhibition activity including thiophene, furan, alkylamines, terminal acetylene, etc. This study can be reference literature for both theoretical and experimental (bio)chemists in several different fields including rational drug design, the process of toxicity prevention, and the discovery of novel inhibitors and catalysts.

Novel bioinformatics quality control metric for next-generation sequencing experiments in the clinical context.

As the use of next-generation sequencing (NGS) for the Mendelian diseases diagnosis is expanding, the performance of this method has to be improved in order to achieve higher quality. Typically, performance measures are considered to be designed in the context of each application and, therefore, account for a spectrum of clinically relevant variants. We present EphaGen, a new computational methodology for bioinformatics quality control (QC). Given a single NGS dataset in BAM format and a pre-compiled VCF-file of targeted clinically relevant variants it associates this dataset with a single arbiter parameter. Intrinsically, EphaGen estimates the probability to miss any variant from the defined spectrum within a particular NGS dataset. Such performance measure virtually resembles the diagnostic sensitivity of given NGS dataset. Here we present case studies of the use of EphaGen in context of BRCA1/2 and CFTR sequencing in a series of 14 runs across 43 blood samples and 504 publically available NGS datasets. EphaGen is superior to conventional bioinformatics metrics such as coverage depth and coverage uniformity. We recommend using this software as a QC step in NGS studies in the clinical context. Availability: https://github.com/m4merg/EphaGen or https://hub.docker.com/r/m4merg/ephagen.

In Vitro and In Vivo Characterization of PLLA-316L Stainless Steel Electromechanical Devices for Bone Tissue Engineering-A Preliminary Study.

Bone injuries represent a major social and financial impairment, commonly requiring surgical intervention due to a limited healing capacity of the tissue, particularly regarding critical-sized defects and non-union fractures. Regenerative medicine with the application of bone implants has been developing in the past decades towards the manufacturing of appropriate devices. This work intended to evaluate medical 316L stainless steel (SS)-based devices covered by a polymer poly (L-lactic acid) (PLLA) coating for bone lesion mechanical and functional support. SS316L devices were subjected to a previously described silanization process, following a three-layer PLLA film coating. Devices were further characterized and evaluated towards their cytocompatibility and osteogenic potential using human dental pulp stem cells, and biocompatibility via subcutaneous implantation in a rat animal model. Results demonstrated PLLA-SS316L devices to present superior in vitro and in vivo outcomes and suggested the PLLA coating to provide osteo-inductive properties to the device. Overall, this work represents a preliminary study on PLLA-SS316L devices' potential towards bone tissue regenerative techniques, showing promising outcomes for bone lesion support.

In search of molecular ions for optical cycling: a difficult road.

Optical cycling, a continuous photon scattering off atoms or molecules, plays a central role in the quantum information science. While optical cycling has been experimentally achieved for many neutral species, few molecular ions have been investigated. We present a systematic theoretical search for diatomic molecular ions suitable for optical cycling using equation-of-motion coupled-cluster methods. Inspired by the electronic structure patterns of laser-cooled neutral molecules, we establish the design principles for molecular ions and explore various possible cationic molecular frameworks. The results show that finding a perfect molecular ion for optical cycling is challenging, yet possible. Among various possible diatomic molecules we suggest several candidates, which require further attention from both theory and experiment: YF+, SiO+, PN+, SiBr+, and BO+.

Towards a rational design of laser-coolable molecules: insights from equation-of-motion coupled-cluster calculations.

Access to cold molecules is critical for quantum information science, design of new sensors, ultracold chemistry, and search of new phenomena. These applications depend on the ability to laser-cool molecules. Rigorous theory and qualitative models can play a central role in narrowing down the vast pool of potential candidates amenable to laser cooling. We report a systematic study of structural and optical properties of alkaline earth metal derivatives in the context of their applicability in laser cooling using equation-of-motion coupled-cluster methods. To rationalize and generalize the results from high-level electronic structure calculations, we develop an effective Hamiltonian model. The model explains the observed trends and suggests new principles for the design of laser-coolable molecules.

Improving Performance of the SMD Solvation Model: Bondi Radii Improve Predicted Aqueous Solvation Free Energies of Ions and pKa Values of Thiols.

Calculation of the solvation free energy of ionic molecules is the principal source of errors in the quantum chemical evaluation of pKa values using implicit polarizable continuum solvent models. One of the important parameters affecting the performance of these models is the choice of atomic radii. Here, we assess the performance of the solvation model based on density (SMD) implicit solvation model employing SMD default radii (SMD) and Bondi radii (SMD-B), a set of empirical atomic radii developed based on the crystallographic data. For a set of 112 ions (60 anions and 52 cations), the SMD-B model showed lower mean unsigned error (MUE) for predicted aqueous solvation free energies (4.0 kcal/mol for anions and 2.4 kcal/mol for cations) compared to the standard SMD model (MUE of 5.0 kcal/mol for anions and 2.9 kcal/mol for cations). In particular, usage of Bondi radii improves the aqueous solvation energies of sulfur-containing ions by >5 kcal/mol compared to the SMD default radii. Indeed, for a set of 45 thiols, the SMD-B model was found to dramatically improve the predicted pKa values, with ∼1 pKa unit mean deviation from the experimental values, compared to ∼7 pKa units mean deviation for the SMD model with the default radii. These findings highlight the importance of the choice of atomic radii on the performance of the implicit solvation models.

From Static to Dynamic: Electron Density of HOMO at Biaryl Linkage Controls the Mechanism of Hole Delocalization.

In order to extend the physical length of hole delocalization in a molecular wire, chromophores of increasing size are often desired. However, the effect of size on the efficacy and mechanism of hole delocalization remains elusive. Here, we employ a model set of biaryls to show that with increasing chromophore size, the mechanism of steady-state hole distribution switches from static delocalization in biaryls with smaller chromophores to dynamic hopping, as exemplified in the largest system, tBuHBC2 (i.e., "superbiphenyl"), which displays a vanishingly small electronic coupling. This important finding is analyzed with the aid of Hückel molecular orbital and Marcus-Hush theories. Our findings will enable the rational design of the novel molecular wires with length-invariant redox/optical properties suitable for long-range charge transfer.

An Electron-Rich Calix[4]arene-Based Receptor with Unprecedented Binding Affinity for Nitric Oxide.

Calixarenes have found widespread application as building blocks for the design and synthesis of functional materials in host-guest chemistry. The ongoing desire to develop a detailed understanding of the nature of NO bonding to multichromophoric π-stacked assemblies led us to develop an electron-rich methoxy derivative of calix[4]arene (3), which we show exists as a single conformer in solution at ambient temperature. Here, we examine the redox properties of this derivative, generate its cation radical (3+. ) using robust chemical oxidants, and determine the relative efficacy of its NO binding in comparison with model calixarenes. We find that 3/3+. is a remarkable receptor for NO+ /NO, with unprecedented binding efficacy. The availability of precise experimental structures of this calixarene derivative and its NO complex, obtained by X-ray crystallography, is critically important both for developing novel functional NO biosensors, and understanding the role of stacked aromatic donors in efficient NO binding, which may have relevance to biological NO transport.

An electron-transfer induced conformational transformation: from non-cofacial "sofa" to cofacial "boat" in cyclotetraveratrylene (CTTV) and formation of charge transfer complexes.

Electro-active polychromophoric assemblies that undergo clam-like electromechanic actuation represent an important class of organic functional materials. Here, we show that the readily available cyclotetraveratrylene (CTTV) undergoes oxidation-induced folding, consistent with interconversion from a non-cofacial "sofa" conformation to a cofacial "boat" conformer. It is found that the non-cofacial "sofa" conformer of CTTV forms stable electron donor-acceptor complexes with chloranil and DDQ. Electron-transfer induced conformational transformation in CTTV provides a framework for the rational design of novel organic functional molecules.