Stem–loop formation drives RNA folding in mechanical unzipping experiments

Accurate knowledge of RNA hybridization is essential for understanding RNA structure and function. Here we mechanically unzip and rezip a 2-kbp RNA hairpin and derive the 10 nearest-neighbor base pair (NNBP) RNA free energies in sodium and magnesium with 0.1 kcal/mol precision using optical tweezers. Notably, force–distance curves (FDCs) exhibit strong irreversible effects with hysteresis and several intermediates, precluding the extraction of the NNBP energies with currently available methods. The combination of a suitable RNA synthesis with a tailored pulling protocol allowed us to obtain the fully reversible FDCs necessary to derive the NNBP energies. We demonstrate the equivalence of sodium and magnesium free-energy salt corrections at the level of individual NNBP. To characterize the irreversibility of the unzipping–rezipping process, we introduce a barrier energy landscape of the stem–loop structures forming along the complementary strands, which compete against the formation of the native hairpin. This landscape correlates with the hysteresis observed along the FDCs. RNA sequence analysis shows that base stacking and base pairing stabilize the stem–loops that kinetically trap the long-lived intermediates observed in the FDC. Stem–loops formation appears as a general mechanism to explain a wide range of behaviors observed in RNA folding.

Rapid Detection of Circulating Tumour DNA using Allele Specific Mini-Loop Mediated Isothermal Amplification

Isothermal amplification is an emerging approach for non-invasive, rapid and cost-effective real-time monitoring of cancer specific mutations through circulating tumour DNA (ctDNA). This study demonstrates a compact allele specific (AS) loop mediated isothermal amplification (LAMP) strategy, termed ‘AS-Mini-LAMP’, modelled using wild type (WT) and mutation specific reactions targeting the estrogen receptor ESR1 c.1138G>C (p.E380Q) missense mutation. Allele selectivity, encoded at the 5’-end of the forward and backward inner primers (FIP and BIP) promotes enhanced selectivity upon self-hybridisation, loop formation and self-primed exponential amplification. Inclusion of unmodified self-stabilising (USS) primers aimed to reduce the likelihood of non-specific allele amplification through competitive inhibition and to enhance reaction velocity through an assisted strand displacement ‘swarm’ priming effect. The two assays were optimised using short synthetic WT and E380Q mutant DNA templates, and subsequently validated to a limit of detection of 500 mutant copies in under 25 minutes in ddPCR-confirmed positive (20.7% variant allele frequency) and negative patient plasma cfDNA samples. These results demonstrate the ability of AS-Mini-LAMP to achieve sensitive and selective amplification of actionable mutations present within plasma ctDNA.

Experimental Study of Hysteresis in Clayey Soils under Compression

The energy dissipation for one cycle of clay soil deformation over the area of ​​a hysteresis loop under conditions of one-dimensional deformation has been experimentally studied. Several series of trials were carried out under different conditions of soil density and moisture and different loading modes. It was established by the experiments that after several cycles of loading and unloading of the samples, the transient process of the closed loop formation ends and certain dependences of stress on deformations are established in the sections of the ascending and descending branches of the hysteresis loop. To determine these dependencies, rheological relations obtained directly from the hysteresis loop by approximating the arcs of its contour have been used. By integrating the approximating rheological dependences along the branches of the loop, the dissipated energy per deformation cycle has been obtained as a function of cyclic deformation amplitude, measured by the area of the hysteresis loop. Experiments on obtaining a hysteresis loop were carried out on a compression device with a cyclic sample. Samples with different states of density and moisture content were produced by consolidating a paste having yield point moisture under different pressures. Several series of experiments have been carried out. In the first series, soil absorption coefficients were derived for different states of density-moisture at different loading rates. In the second series, three types of clayish soil (clay, loam, sandy loam) were studied. Dissipation coefficients have been found out for the indicated soils. In the third series, three types of clay soil were tested under different conditions of density and moisture. The dissipation coefficients have been obtained. In the fourth series, the dependences of the absorption coefficient on the amplitude value of the cyclic stress for three types of clay soil were disclosed. It was found that a change in the loading rate within the range from 0.05 MPa to 0.2 MPa does not lead to the significant change in the absorption coefficient, the increase in the number of clay fractions in the sample leads to an increase in the absorption coefficient, a change in the amplitude of cyclic loading (in the indicated range of change) does not affect the absorption coefficient.

Proximity labeling identifies a repertoire of site-specific R-loop modulators

R-loops are three-stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains. One of the most consistently enriched proteins was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP resolves R-loops in vitro and that it is necessary to suppress R-loops in vivo at its genomic targets. Furthermore, deletion of the ADNP homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets, and compromises neuronal differentiation. Notably, patient-derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci

Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to β-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated for the first time that this can occur through multiple D-loop formation from one donor DNA molecule or by the uptake of multiple DNA fragments. We also show that the final minimum inhibitory concentration differs depending on recipient genome, and the sampled fitness landscape. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we show that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake matters. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.

RNA binding protein RBM3 augments kissing loop formation with lncRNAs to enhance translational control

Background and Aims: Translational regulation involve the coordinated actions of RNA binding proteins (RBPs) and non-coding RNAs. For efficient translation, the mRNA needs to be circularized. While RNA binding proteins and translation factors have been shown to regulate the circularization, the role of lncRNAs in the process is not yet defined. Methods: We first performed RNA-seq and RNA-immunoprecipitation coupled-Seq (RIP-Seq) to identify differentially expressed lncRNA and mRNA in RBM3 overexpressing cell lines. We manipulated lncRNA expression in the cells and determined effects on gene expression and cell viability and motility. The studies were confirmed in vivo in intestine specific RBM3 transgenic and RBM3 knockout mouse models. Results: In comparing the RNA-Seq and RIP-Seq datasets, we identified increased expression of lncRNA LSAMP-3 and Flii-1 that bind to RBM3. In addition, there was an increase in expression of epithelial mesenchymal transition and angiogenesis markers following RBM3 overexpression. Moreover, modeling studies suggest that these lncRNAs formed kissing-loop interactions on target mRNAs including transcripts that encode epithelial mesenchymal transition and angiogenesis. While RBM3 transgenic mice showed increased LSAMP-3 and Flii-1, this was reduced in the RBM3 knockout mice. Also, RBM3 overexpression increased tumor xenograft growth, which was suppressed by knockdown of the lncRNAs. Also, knockdown of endogenous RBM3 specifically in the intestine suppressed azoxymethane-dextran sodium sulfate driven colitis-associated cancers, with a corresponding reduction in the expression of lncRNAs and transcripts that encode epithelial mesenchymal transition and angiogenesis. Conclusion: We propose that RBPs such as RBM3 mediate their function through regulatory lncRNAs that enable circularization to control translation.

TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA

The telomere specific shelterin complex, which includes TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, prevents spurious recognition of telomeres as double-strand DNA breaks and regulates telomerase and DNA repair activities at telomeres. TIN2 is a key component of the shelterin complex that directly interacts with TRF1, TRF2 and TPP1. In vivo, the large majority of TRF1 and TRF2 are in complex with TIN2 but without TPP1 and POT1. Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, previous cell-based assays only provide information on downstream effects after the loss of TRF1/TRF2 and TIN2. Here, we investigated DNA structures promoted by TRF2–TIN2 using single-molecule imaging platforms, including tracking of compaction of long mouse telomeric DNA using fluorescence imaging, atomic force microscopy (AFM) imaging of protein–DNA structures, and monitoring of DNA–DNA and DNA–RNA bridging using the DNA tightrope assay. These techniques enabled us to uncover previously unknown unique activities of TIN2. TIN2S and TIN2L isoforms facilitate TRF2-mediated telomeric DNA compaction (cis-interactions), dsDNA–dsDNA, dsDNA–ssDNA and dsDNA–ssRNA bridging (trans-interactions). Furthermore, TIN2 facilitates TRF2-mediated T-loop formation. We propose a molecular model in which TIN2 functions as an architectural protein to promote TRF2-mediated trans and cis higher-order nucleic acid structures at telomeres.

Cohesin modulates DNA replication to preserve genome integrity

Cohesin participates in loop formation by extruding DNA fibers from its ring-shaped structure. Cohesin dysfunction eliminates chromatin loops but only causes modest transcription perturbation, which cannot fully explain the frequently observed mutations of cohesin in various cancers. Here, we found that DNA replication initiates at more than one thousand extra dormant origins after acute depletion of RAD21, a core subunit of cohesin, resulting in earlier replicating timing at approximately 30% of the human genomic regions. In contrast, CTCF is dispensable for suppressing the early firing of dormant origins that are distributed away from the loop boundaries. Furthermore, greatly elevated levels of gross DNA breaks and genome-wide chromosomal translocations arise in RAD21-depleted cells, accompanied by dysregulated replication timing at dozens of hotspot genes. Thus, we conclude that cohesin coordinates DNA replication initiation to ensure proper replication timing and safeguards genome integrity.

Increased replication stress and R-loop accumulation in EGFRvIII expressing glioblastoma present new therapeutic opportunities

Background The oncogene epidermal growth factor receptor variant III (EGFRvIII) is expressed in approximately one third of all glioblastoma (GBM). So far it is not clear if EGFRvIII expression induces replication stress in GBM cells, which might serve as a therapeutical target. Methods Isogenetic EGFRvIII- and EGFRvIII+ cell lines with endogenous EGFRvIII expression were used. Markers of oncogenic and replication stress such as γH2AX, RPA, 53BP1, ATR and Chk1 were analysed using western blot, immunofluorescence and flow cytometry. The DNA fiber assay was performed to analyse replication, transcription was measured by incorporation of EU and genomic instability was investigated by micronuclei and CGH-Array analysis. Immunohistochemistry staining was used to detect replication stress markers and R-loops in human GBM samples. Results EGFRvIII+ cells exhibit an activated replication stress response, increased spontaneous DNA damage, elevated levels of single stranded DNA and reduced DNA replication velocity, which are all indicative characteristics for replication stress. Furthermore, we show here that EGFRvIII expression is linked to increased genomic instability. EGFRvIII expressing cells display elevated RNA synthesis and R-loop formation, which could also be confirmed in EGFRvIII positive GBM patient samples. Targeting replication stress by irinotecan resulted in increased sensitivity of EGFRvIII+ cells. Conclusion This study demonstrates that EGFRvIII expression is associated with increased replication stress, R-loop accumulation and genomic instability. This might contribute to intratumoral heterogeneity but may also be exploited for individualized therapy approaches.