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The coexistence of transposons with their hosts depends largely on transposition levels being tightly regulated to limit the mutagenic burden associated with frequent transposition. For 'DNA-based' (class II) bacterial transposons there is growing evidence that regulation through small noncoding RNAs and/or the RNA-binding protein Hfq are prominent mechanisms of defense against transposition. Recent transcriptomics analyses have identified many new cases of antisense RNAs (asRNA) that potentially could regulate the expression of transposon-encoded genes giving the impression that asRNA regulation of DNA-based transposons is much more frequent than previously thought. Hfq is a highly conserved bacterial protein that plays a central role in posttranscriptional gene regulation and stress response pathways in many bacteria. Three different mechanisms for Hfq-directed control of bacterial transposons have been identified to date highlighting the versatility of this protein as a regulator of bacterial transposons. There is also evidence emerging that some DNA-based transposons encode RNAs that could regulate expression of host genes. In the case of IS200, which appears to have lost its ability to transpose, contributing a regulatory RNA to its host could account for the persistence of this mobile element in a wide range of bacterial species. It remains to be seen how prevalent these transposon-encoded RNA regulators are, but given the relatively large amount of intragenic transcription in bacterial genomes, it would not be surprising if new examples are forthcoming. WIREs RNA 2016, 7:382-398. doi: 10.1002/wrna.1341 For further resources related to this article, please visit the WIREs website., (© 2016 Wiley Periodicals, Inc.)

IS200 is found throughout Enterobacteriaceae and transposes at a notoriously low frequency. In addition to the transposase protein (TnpA), IS200 encodes an uncharacterized Hfq-binding sRNA that is encoded opposite to the tnpA 5'UTR. In the current work we asked if this sRNA represses tnpA expression. We show here that the IS200 sRNA (named art200 for antisense regulator of transposase IS200) basepairs with tnpA to inhibit translation initiation. Unexpectedly, art200-tnpA pairing is limited to 40 bp, despite 90 nt of perfect complementarity. Additionally, we show that Hfq and RNA secondary structure in the tnpA 5'UTR each repress tnpA expression in an art200-independent manner. Finally, we show that disrupting translational control of tnpA expression leads to increased IS200 transposition in E. coli. The current work provides new mechanistic insight into why IS200 transposition is so strongly suppressed. The possibility of art200 acting in trans to regulate a yet-unidentified target is discussed as well as potential applications of the IS200 system for designing novel riboregulators., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The study of the bacterial transposons Tn10 and Tn5 has provided a wealth of information regarding steps in nonreplicative DNA transposition, transpososome dynamics and structure, as well as mechanisms employed to regulate transposition. The focus of ongoing research on these transposons is mainly on host regulation and the use of the Tn10 antisense system as a platform to develop riboregulators for applications in synthetic biology. Over the past decade two new regulators of both Tn10 and Tn5 transposition have been identified, namely H-NS and Hfq proteins. These are both global regulators of gene expression in enteric bacteria with functions linked to stress-response pathways and virulence and potentially could link the Tn10 and Tn5 systems (and thus the transfer of antibiotic resistance genes) to environmental cues. Work summarized here is consistent with the H-NS protein working directly on transposition complexes to upregulate both Tn10 and Tn5 transposition. In contrast, evidence is discussed that is consistent with Hfq working at the level of transposase expression to downregulate both systems. With regard to Tn10 and synthetic biology, some recent work that incorporates the Tn10 antisense RNA into both transcriptional and translational riboswitches is summarized.

RNA footprinting and structure probing techniques are used to characterize the interaction between RNA-binding proteins and RNAs in vitro. Hydroxyl radical footprinting results in the identification of protein binding site(s) in an RNA. Ribonuclease (RNase) structure probing is a complementary technique that also provides information about protein binding sites, as well as RNA structure and possible protein-directed RNA remodeling. Here we provide a comprehensive protocol for studying the interaction between Hfq and an mRNA or sRNA of interest using a combination of RNase A, T1, and V1 as well as hydroxyl radical footprinting techniques. Detailed protocols for in vitro synthesis of (32)P-labeled RNA; formation of Hfq:RNA binary complex(es), RNase, and hydroxyl radical footprinting; preparation and running of sequencing gels; and data analysis are provided.

Background and Objective: To date, there have been few published studies examining the relationship between arteriovenous malformation (AVM) angioarchitecture and hemorrhagic presentation among children with cerebral AVMs. This study examines this relationship in this unique population, in whom symptomatic presentation of cerebral AVM is the norm rather than the exception., Methods: A cohort of children with AVMs from 2000 to 2011 were included. Predictors studied included patient age, gender and angioarchitectural features, including AVM location, nidus size and morphology, venous drainage, presence of venous outflow lesions and associated aneurysms. Predictors of hemorrhagic presentation were assessed using multivariate logistic regression., Results: 135 children (70 males, mean age 10.1 years) were included. 86/135 (63.7%) children presented with hemorrhage, 18 (13.3%) with seizures, 17 (12.6%) with headaches or neurological deficits and 14 (10.4%) were asymptomatic. AVM location, morphology and the presence of associated aneurysm, venous ectasia, draining vein stenosis and single draining vein were not significantly associated factors. After multivariate analysis, AVM size (OR 0.57, 95% CI 0.43 to 0.77; p<0.01), exclusive deep venous drainage (OR 4.94, 95% CI 1.30 to 18.8; p=0.02) and infratentorial location (OR 9.94, 95% CI 1.71 to 51.76; p=0.01) were independently associated with hemorrhagic presentation., Conclusion: Smaller AVM size, exclusive deep venous drainage and infratentorial location are specific angioarchitectural factors independently associated with initial hemorrhagic presentation in children with AVMs.

The biophysical and biological characterization of 8,13-diethyl-6-methylquino[4,3,2-k]lacridinium iodide (6) is reported. The compound binds to DNA, as measured by UV, fluorescence, and circular dichroism studies, and stabilizes the double helix and higher order DNA structures (DNA triplexes and quadruplexes) against thermal denaturation. Unlike many DNA ligands, (6) shows no specificity for binding to specific base pair combinations and does not inhibit topoisomerase I (topo I) or topo II activity. Furthermore, the biological fingerprint elicited by (6) in in vitro evaluations does not compare with clinical agents of the topo II inhibition class. The compound provokes cell cycle arrest in response to DNA damage and the biological sequelae are dependent on the p53 status of the cell line. DNA damage by (6) upregulates p53 and p21(CIP/WAF1) proteins. The unusual structure of (6) and its ease of synthesis in a "one-pot" reaction are features that are being exploited in the design and development of a new series of G-quadruplex stabilizing telomerase inhibitors. However, although the second-generation compounds that resulted from (6) present strong telomerase inhibition, (6) in itself presents yet a different mode of action, with a strong preference for triplex DNA, sequences often found in a number of genes.