Common fragile sites (CFSs) are genomic regions prone to breakage under replication stress conditions recurrently rearranged in cancer. Many CFSs are enriched with AT-dinucleotide rich sequences (AT-DRSs) which have the potential to form stable secondary structures upon unwinding the double helix during DNA replication. These stable structures can potentially perturb DNA replication progression, leading to genomic instability. Using site-specific targeting system, we show that targeted integration of a 3.4 kb AT-DRS derived from the human CFS FRA16C into a chromosomally stable region within the human genome is able to drive fragile site formation under conditions of replication stress. Analysis of >1300 X chromosomes integrated with the 3.4 kb AT-DRS revealed recurrent gaps and breaks at the integration site. DNA sequences derived from the integrated AT-DRS showed in vitro a significantly increased tendency to fold into branched secondary structures, supporting the predicted mechanism of instability. Our findings clearly indicate that intrinsic DNA features, such as complexed repeated sequence motifs, predispose the human genome to chromosomal instability.

Small non-coding RNAs (sRNAs) regulate numerous cellular processes in all domains of life. Several approaches have been developed to identify them from RNA-seq data, which are efficient for eukaryotic sRNAs but remain inaccurate for the longer and highly structured bacterial sRNAs. We present APERO, a new algorithm to detect small transcripts from paired-end bacterial RNA-seq data. In contrast to previous approaches that start from the read coverage distribution, APERO analyzes boundaries of individual sequenced fragments to infer the 5' and 3' ends of all transcripts. Since sRNAs are about the same size as individual fragments (50-350 nucleotides), this algorithm provides a significantly higher accuracy and robustness, e.g., with respect to spontaneous internal breaking sites. To demonstrate this improvement, we develop a comparative assessment on datasets from Escherichia coli and Salmonella enterica, based on experimentally validated sRNAs. We also identify the small transcript repertoire of Dickeya dadantii including putative intergenic RNAs, 5' UTR or 3' UTR-derived RNA products and antisense RNAs. Comparisons to annotations as well as RACE-PCR experimental data confirm the precision of the detected transcripts. Altogether, APERO outperforms all existing methods in terms of sRNA detection and boundary precision, which is crucial for comprehensive genome annotations. It is freely available as an open source R package on https://github.com/Simon-Leonard/APERO.

Dynamic and reversible RNA modifications such as N6-methyladenosine (m6A) can play important roles in regulating messenger RNA (mRNA) splicing, export, stability and translation. Defective mRNA modification through altered expression of the methyltransferase and/or demethylases results in developmental defects and cancer progression. Identifying modified mRNAs, annotating the distribution of modification sites across the mRNA, as well as characterizing and comparing other modification features are essential for studying the function and elucidating the mechanism of mRNA modifications. Several methods including methylated RNA immunoprecipitation and sequencing (MeRIP-seq) are available for the detection of mRNA modifications. However, a convenient and comprehensive tool to annotate diverse kinds of mRNA modifications in different species is lacking. Here, we developed RNAmod (https://bioinformatics.sc.cn/RNAmod), an interactive, one-stop, web-based platform for the automated analysis, annotation, and visualization of mRNA modifications in 21 species. RNAmod provides intuitive interfaces to show outputs including the distribution of RNA modifications, modification coverage for different gene features, functional annotation of modified mRNAs, and comparisons between different groups or specific gene sets. Furthermore, sites of known RNA modification, as well as binding site data for hundreds of RNA-binding proteins (RBPs) are integrated in RNAmod to help users compare their modification data with known modifications and to explore the relationship with the binding sites of known RBPs. RNAmod is freely available and meets the emerging need for a convenient and comprehensive analysis tool for the fast-developing RNA modification field.

AntimiR is an antisense oligonucleotide that has been developed to silence microRNA (miRNA) for the treatment of intractable diseases. Enhancement of its in vivo efficacy and improvement of its toxicity are highly desirable but remain challenging. We here design heteroduplex oligonucleotide (HDO)-antimiR as a new technology comprising an antimiR and its complementary RNA. HDO-antimiR binds targeted miRNA in vivo more efficiently by 12-fold than the parent single-stranded antimiR. HDO-antimiR also produced enhanced phenotypic effects in mice with upregulated expression of miRNA-targeting messenger RNAs. In addition, we demonstrated that the enhanced potency of HDO-antimiR was not explained by its bio-stability or delivery to the targeted cell, but reflected an improved intracellular potency. Our findings provide new insights into biology of miRNA silencing by double-stranded oligonucleotides and support the in vivo potential of this technology based on a new class of for the treatment of miRNA-related diseases.

The intron-lariat spliceosome (ILS) complex is highly conserved among eukaryotes, and its disassembly marks the end of a canonical splicing cycle. In this study, we show that two conserved disassembly factors of the ILS complex, Increased Level of Polyploidy1-1D (ILP1) and NTC-Related protein 1 (NTR1), positively regulate microRNA (miRNA) biogenesis by facilitating transcriptional elongation of MIRNA (MIR) genes in Arabidopsis thaliana. ILP1 and NTR1 formed a stable complex and co-regulated alternative splicing of more than a hundred genes across the Arabidopsis genome, including some primary transcripts of miRNAs (pri-miRNAs). Intriguingly, pri-miRNAs, regardless of having introns or not, were globally down-regulated when the ILP1 or NTR1 function was compromised. ILP1 and NTR1 interacted with core miRNA processing proteins Dicer-like 1 and Serrate, and were required for proper RNA polymerase II occupancy at elongated regions of MIR chromatin, without affecting either MIR promoter activity or pri-miRNA decay. Our results provide further insights into the regulatory role of spliceosomal machineries in the biogenesis of miRNAs.

Nucleosomal DNA sequences generally follow a well-known pattern with ∼10-bp periodic WW (where W is A or T) dinucleotides that oscillate in phase with each other and out of phase with SS (where S is G or C) dinucleotides. However, nucleosomes with other DNA patterns have not been systematically analyzed. Here, we focus on an opposite pattern, namely anti-WW/SS pattern, in which WW dinucleotides preferentially occur at DNA sites that bend into major grooves and SS (where S is G or C) dinucleotides are often found at sites that bend into minor grooves. Nucleosomes with the anti-WW/SS pattern are widespread and exhibit a species- and context-specific distribution in eukaryotic genomes. Unlike non-mammals (yeast, nematode and fly), there is a positive correlation between the enrichment of anti-WW/SS nucleosomes and RNA Pol II transcriptional levels in mammals (mouse and human). Interestingly, such enrichment is not due to underlying DNA sequence. In addition, chromatin remodeling complexes have an impact on the abundance but not on the distribution of anti-WW/SS nucleosomes in yeast. Our data reveal distinct roles of cis- and trans-acting factors in the rotational positioning of nucleosomes between non-mammals and mammals. Implications of the anti-WW/SS sequence pattern for RNA Pol II transcription are discussed.

DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes' local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.

The nematode Caenorhabditis elegans contains several types of endogenous small interfering RNAs (endo-siRNAs) produced by RNA-dependent RNA polymerase (RdRP) complexes. Both 'silencing' siRNAs bound by Worm-specific Argonautes (WAGO) and 'activating' siRNAs bound by the CSR-1 Argonaute require the DRH-3 helicase, an RdRP component. Here, we show that, in the drh-3(ne4253) mutant deficient in RdRP-produced secondary endo-siRNAs, the silencing histone mark H3K9me3 is largely depleted, whereas in the csr-1 partially rescued null mutant strain (WM193), this mark is ectopically deposited on CSR-1 target genes. Moreover, we observe ectopic H3K9me3 at enhancer elements and an increased number of small RNAs that match enhancers in both drh-3 and csr-1 mutants. Finally, we detect accumulation of H3K27me3 at highly expressed genes in the drh-3(ne4253) mutant, which correlates with their reduced transcription. Our study shows that when abundant RdRP-produced siRNAs are depleted, there is ectopic elevation of noncoding RNAs linked to sites with increased silencing chromatin marks. Moreover, our results suggest that enhancer small RNAs may guide local H3K9 methylation.

Terminal deoxynucleotidyl transferase (TdT), which mediates template-independent polymerization with low specificity for nucleotides, has been used for nucleotide extension of DNA oligomers. One concern is that it is difficult to control the number of incorporated nucleotides, which is a limitation on the use of TdT for single-nucleotide incorporation of DNA oligomers. Herein, we uncovered an interesting inhibitory effect on TdT when ribonucleotide substrates (rNTPs) were employed in a borate buffer. On the basis of unique inhibitory effects of the ribonucleotide-borate complex, we developed a novel enzymatic method for single-nucleotide incorporation of a DNA oligomer with a modified rNTP by TdT. Single-nucleotide incorporation of a DNA oligomer with various modified rNTPs containing an oxanine, biotin, aminoallyl or N6-propargyl group was achieved with a high yield. The 'TdT in rNTP-borate' method is quite simple and efficient for preparing a single-nucleotide modified DNA oligomer, which is useful for the design of functional DNA-based systems.

Methyl-5-uridine (m5U) is one the most abundant non-canonical bases present in cellular RNA, and in yeast is found at position U54 of tRNAs where modification is catalysed by the methyltransferase Trm2. Although the mammalian enzymes that catalyse m5U formation are yet to be identified via experimental evidence, based on sequence homology to Trm2, two candidates currently exist, TRMT2A and TRMT2B. Here we developed a genome-wide single-nucleotide resolution mapping method, Fluorouracil-Induced-Catalytic-Crosslinking-Sequencing (FICC-Seq), in order to identify the relevant enzymatic targets. We demonstrate that TRMT2A is responsible for the majority of m5U present in human RNA, and that it commonly targets U54 of cytosolic tRNAs. By comparison to current methods, we show that FICC-Seq is a particularly robust method for accurate and reliable detection of relevant enzymatic target sites. Our associated finding of extensive irreversible TRMT2A-tRNA crosslinking in vivo following 5-Fluorouracil exposure is also intriguing, as it suggests a tangible mechanism for a previously suspected RNA-dependent route of Fluorouracil-mediated cytotoxicity.

Restriction-modification (R-M) systems represent an effective mechanism of defence against invading bacteriophages, and are widely spread among bacteria and archaea. In acquiring a Type II R-M system via horizontal gene transfer, the new hosts become more resistant to phage infection, through the action of a restriction endonuclease (REase), which recognizes and cleaves specific target DNAs. To protect the host cell's DNA, there is also a methyltransferase (MTase), which prevents DNA cleavage by the cognate REase. In some R-M systems, the host also accepts a cis-acting transcription factor (C protein), which regulates the counteracting activities of REase and MTase to avoid host self-restriction. Our study characterized the unexpected phenotype of Escherichia coli cells, which manifested as extensive cell filamentation triggered by acquiring the Csp231I R-M system from Citrobacter sp. Surprisingly, we found that the cell morphology defect was solely dependent on the C regulator. Our transcriptome analysis supported by in vivo and in vitro assays showed that C protein directly silenced the expression of the RacR repressor to affect the Rac prophage-related genes. The rac locus ydaST genes, when derepressed, exerted a toxicity indicated by cell filamentation through an unknown mechanism. These results provide an apparent example of transcription factor cross-talk, which can have significant consequences for the host, and may represent a constraint on lateral gene transfer.

A primary challenge in the analysis of RNA-seq data is to identify differentially expressed genes or transcripts while controlling for technical biases. Ideally, a statistical testing procedure should incorporate the inherent uncertainty of the abundance estimates arising from the quantification step. Most popular methods for RNA-seq differential expression analysis fit a parametric model to the counts for each gene or transcript, and a subset of methods can incorporate uncertainty. Previous work has shown that nonparametric models for RNA-seq differential expression may have better control of the false discovery rate, and adapt well to new data types without requiring reformulation of a parametric model. Existing nonparametric models do not take into account inferential uncertainty, leading to an inflated false discovery rate, in particular at the transcript level. We propose a nonparametric model for differential expression analysis using inferential replicate counts, extending the existing SAMseq method to account for inferential uncertainty. We compare our method, Swish, with popular differential expression analysis methods. Swish has improved control of the false discovery rate, in particular for transcripts with high inferential uncertainty. We apply Swish to a single-cell RNA-seq dataset, assessing differential expression between sub-populations of cells, and compare its performance to the Wilcoxon test.

CpG islands (CGIs) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression.

Translation regulation plays an important role in eukaryotic gene expression. Upstream open reading frames (uORFs) are potent regulatory elements located in 5' mRNA transcript leaders. Translation of uORFs usually inhibit the translation of downstream main open reading frames, but some enhance expression. While a minority of uORFs encode conserved functional peptides, the coding regions of most uORFs are not conserved. Thus, the importance of uORF coding sequences on their regulatory functions remains largely unknown. We investigated the impact of an uORF coding region on gene regulation by assaying the functions of thousands of variants in the yeast YAP1 uORF. Varying uORF codons resulted in a wide range of functions, including repressing and enhancing expression of the downstream ORF. The presence of rare codons resulted in the most inhibitory YAP1 uORF variants. Inhibitory functions of such uORFs were abrogated by overexpression of complementary tRNA. Finally, regression analysis of our results indicated that both codon identity and position impact uORF function. Our results support a model in which a uORF coding sequence impacts its regulatory functions by altering the speed of uORF translation.

Plus-strand RNA viruses can accumulate viral RNA degradation products during infections. Some of these decay intermediates are generated by the cytosolic 5'-to-3' exoribonuclease Xrn1 (mammals and yeast) or Xrn4 (plants) and are formed when the enzyme stalls on substrate RNAs upon encountering inhibitory RNA structures. Many Xrn-generated RNAs correspond to 3'-terminal segments within the 3'-UTR of viral genomes and perform important functions during infections. Here we have investigated a 3'-terminal small viral RNA (svRNA) generated by Xrn during infections with Tobacco necrosis virus-D (family Tombusviridae). Our results indicate that (i) unlike known stalling RNA structures that are compact and modular, the TNV-D structure encompasses the entire 212 nt of the svRNA and is not functionally transposable, (ii) at least two tertiary interactions within the RNA structure are required for effective Xrn blocking and (iii) most of the svRNA generated in infections is derived from viral polymerase-generated subgenomic mRNA1. In vitro and in vivo analyses allowed for inferences on roles for the svRNA. Our findings provide a new and distinct addition to the growing list of Xrn-resistant viral RNAs and stalling structures found associated with different plant and animal RNA viruses.

When double-strand breaks are introduced in a genome by CRISPR they are repaired either by non-homologous end joining (NHEJ), which often results in insertions or deletions (indels), or by homology-directed repair (HDR), which allows precise nucleotide substitutions to be introduced if a donor oligonucleotide is provided. Because NHEJ is more efficient than HDR, the frequency with which precise genome editing can be achieved is so low that simultaneous editing of more than one gene has hitherto not been possible. Here, we introduced a mutation in the human PRKDC gene that eliminates the kinase activity of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). This results in an increase in HDR irrespective of cell type and CRISPR enzyme used, sometimes allowing 87% of chromosomes in a population of cells to be precisely edited. It also allows for precise editing of up to four genes simultaneously (8 chromosomes) in the same cell. Transient inhibition of DNA-PKcs by the kinase inhibitor M3814 is similarly able to enhance precise genome editing.

The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

Long Interspersed Elements (LINEs), also known as non-LTR retrotransposons, encode a multifunctional protein that reverse transcribes its mRNA into DNA at the site of insertion by target primed reverse transcription. The second half of the integration reaction remains very poorly understood. Second-strand DNA cleavage and second-strand DNA synthesis were investigated in vitro using purified components from a site-specific restriction-like endonuclease (RLE) bearing LINE. DNA structure was shown to be a critical component of second-strand DNA cleavage. A hitherto unknown and unexplored integration intermediate, an open '4-way' DNA junction, was recognized by the element protein and cleaved in a Holliday junction resolvase-like reaction. Cleavage of the 4-way junction resulted in a natural primer-template pairing used for second-strand DNA synthesis. A new model for RLE LINE integration is presented.

Some viruses package dsDNA together with large amounts of positively charged proteins, thought to help condense the genome inside the capsid with no evidence. Further, this role is not clear because these viruses have typically lower packing fractions than viruses encapsidating naked dsDNA. In addition, it has recently been shown that the major adenovirus condensing protein (polypeptide VII) is dispensable for genome encapsidation. Here, we study the morphology and mechanics of adenovirus particles with (Ad5-wt) and without (Ad5-VII-) protein VII. Ad5-VII- particles are stiffer than Ad5-wt, but DNA-counterions revert this difference, indicating that VII screens repulsive DNA-DNA interactions. Consequently, its absence results in increased internal pressure. The core is slightly more ordered in the absence of VII and diffuses faster out of Ad5-VII- than Ad5-wt fractured particles. In Ad5-wt unpacked cores, dsDNA associates in bundles interspersed with VII-DNA clusters. These results indicate that protein VII condenses the adenovirus genome by combining direct clustering and promotion of bridging by other core proteins. This condensation modulates the virion internal pressure and DNA release from disrupted particles, which could be crucial to keep the genome protected inside the semi-disrupted capsid while traveling to the nuclear pore.

Natural products represent a rich reservoir of small molecule drug candidates utilized as antimicrobial drugs, anticancer therapies, and immunomodulatory agents. These molecules are microbial secondary metabolites synthesized by co-localized genes termed Biosynthetic Gene Clusters (BGCs). The increase in full microbial genomes and similar resources has led to development of BGC prediction algorithms, although their precision and ability to identify novel BGC classes could be improved. Here we present a deep learning strategy (DeepBGC) that offers reduced false positive rates in BGC identification and an improved ability to extrapolate and identify novel BGC classes compared to existing machine-learning tools. We supplemented this with random forest classifiers that accurately predicted BGC product classes and potential chemical activity. Application of DeepBGC to bacterial genomes uncovered previously undetectable putative BGCs that may code for natural products with novel biologic activities. The improved accuracy and classification ability of DeepBGC represents a major addition to in-silico BGC identification.