Synthetic riboswitches for the analysis of tRNA processing by eukaryotic RNase P enzymes.

Removal of the 5'-leader region is an essential step in the maturation of tRNA molecules in all domains of life. This reaction is catalyzed by various RNase P activities, ranging from ribonucleoproteins with ribozyme activity to protein-only forms. In Escherichia coli, the efficiency of RNase P-mediated cleavage can be controlled by computationally designed riboswitch elements in a ligand-dependent way, where the 5'-leader sequence of a tRNA precursor is either sequestered in a hairpin structure or presented as a single-stranded region accessible for maturation. In the presented work, the regulatory potential of such artificial constructs is tested on different forms of eukaryotic RNase P enzymes-two protein-only RNase P enzymes (PRORP1 and PRORP2) from Arabidopsis thaliana and the ribonucleoprotein of Homo sapiens The PRORP enzymes were analyzed in vitro as well as in vivo in a bacterial RNase P complementation system. We also tested in HEK293T cells whether the riboswitches remain functional with human nuclear RNase P. While the regulatory principle of the synthetic riboswitches applies for all tested RNase P enzymes, the results also show differences in the substrate requirements of the individual enzyme versions. Hence, such designed RNase P riboswitches represent a novel tool to investigate the impact of the structural composition of the 5'-leader on substrate recognition by different types of RNase P enzymes.

Ligand-dependent tRNA processing by a rationally designed RNase P riboswitch.

We describe a synthetic riboswitch element that implements a regulatory principle which directly addresses an essential tRNA maturation step. Constructed using a rational in silico design approach, this riboswitch regulates RNase P-catalyzed tRNA 5'-processing by either sequestering or exposing the single-stranded 5'-leader region of the tRNA precursor in response to a ligand. A single base pair in the 5'-leader defines the regulatory potential of the riboswitch both in vitro and in vivo. Our data provide proof for prior postulates on the importance of the structure of the leader region for tRNA maturation. We demonstrate that computational predictions of ligand-dependent structural rearrangements can address individual maturation steps of stable non-coding RNAs, thus making them amenable as promising target for regulatory devices that can be used as functional building blocks in synthetic biology.

Rhodol-based thallium sensors for cellular imaging of potassium channel activity.

Thallium (Tl+) flux assays enable imaging of potassium (K+) channel activity in cells and tissues by exploiting the permeability of K+ channels to Tl+ coupled with a fluorescent Tl+ sensitive dye. Common Tl+ sensing dyes utilize fluorescein as the fluorophore though fluorescein exhibits certain undesirable properties in these assays including short excitation wavelengths and pH sensitivity. To overcome these drawbacks, the replacement of fluorescein with rhodols was investigated. A library of 13 rhodol-based Tl+ sensors was synthesized and their properties and performance in Tl+ flux assays evaluated. The dimethyl rhodol Tl+ sensor emerged as the best of the series and performed comparably to fluorescein-based sensors while demonstrating greater pH tolerance in the physiological range and excitation and emission spectra 30 nm red-shifted from fluorescein.

RNA Design Principles for Riboswitches that Regulate RNase P-Mediated tRNA Processing.

Riboswitches are an attractive target for the directed design of RNA-based regulators by in silico prediction. These noncoding RNA elements consist of an aptamer platform for the highly selective ligand recognition and an expression platform which controls gene activity typically at the level of transcription or translation. In previous work, we could successfully apply RNA folding prediction to implement a new riboswitch mechanism regulating processing of a tRNA by RNase P. In this contribution, we present detailed information about our pipeline consisting of in silico design combined with the biochemical analysis for the verification of the implemented mechanism. Furthermore, we discuss the applicability of the presented biochemical in vivo and in vitro methods for the characterization of other artificial riboswitches.

Active RhoA Exerts an Inhibitory Effect on the Homeostasis and Angiogenic Capacity of Human Endothelial Cells.

Background The small GTPase RhoA (Ras homolog gene family, member A) regulates a variety of cellular processes, including cell motility, proliferation, survival, and permeability. In addition, there are reports indicating that RhoA-ROCK (rho associated coiled-coil containing protein kinase) activation is essential for VEGF (vascular endothelial growth factor)-mediated angiogenesis, whereas other work suggests VEGF-antagonistic effects of the RhoA-ROCK axis. Methods and Results To elucidate this issue, we examined human umbilical vein endothelial cells and human coronary artery endothelial cells after stable overexpression (lentiviral transduction) of constitutively active (G14V/Q63L), dominant-negative (T19N), or wild-type RhoA using a series of in vitro angiogenesis assays (proliferation, migration, tube formation, angiogenic sprouting, endothelial cell viability) and a human umbilical vein endothelial cells xenograft assay in immune-incompetent NOD scid gamma mice in vivo. Here, we report that expression of active and wild-type RhoA but not dominant-negative RhoA significantly inhibited endothelial cell proliferation, migration, tube formation, and angiogenic sprouting in vitro. Moreover, active RhoA increased endothelial cell death in vitro and decreased human umbilical vein endothelial cell-related angiogenesis in vivo. Inhibition of RhoA by C3 transferase antagonized the inhibitory effects of RhoA and strongly enhanced VEGF-induced angiogenic sprouting in control-treated cells. In contrast, inhibition of RhoA effectors ROCK1/2 and LIMK1/2 (LIM domain kinase 1/2) did not significantly affect RhoA-related effects, but increased angiogenic sprouting and migration of control-treated cells. In agreement with these data, VEGF did not activate RhoA in human umbilical vein endothelial cells as measured by a Förster resonance energy transfer-based biosensor. Furthermore, global transcriptome and subsequent bioinformatic gene ontology enrichment analyses revealed that constitutively active RhoA induced a differentially expressed gene pattern that was enriched for gene ontology biological process terms associated with mitotic nuclear division, cell proliferation, cell motility, and cell adhesion, which included a significant decrease in VEGFR-2 (vascular endothelial growth factor receptor 2) and NOS3 (nitric oxide synthase 3) expression. Conclusions Our data demonstrate that increased RhoA activity has the potential to trigger endothelial dysfunction and antiangiogenic effects independently of its well-characterized downstream effectors ROCK and LIMK.

Evaluation of ß-d-glucuronidase and particle-size distribution for microbiological water quality monitoring in Northern Vietnam.

In many karst regions in developing countries, the populations often suffer from poor microbial water quality and are frequently exposed to bacterial pathogens. The high variability of water quality requires rapid assays, but the conventional cultivation-based analysis of fecal indicator bacteria, such as Escherichia coli (E. coli), is very time-consuming. In this respect, the measurement of the enzymatic activity of E. coli could prove to be a valuable tool for water quality monitoring. A mobile automated prototype device was used for the investigation of ß-d-glucuronidase (GLUC) activity at a remote karst spring, connected to a sinking surface stream, in Northern Vietnam. To assess the relationship between GLUC activity, discharge dynamics and contamination patterns, multiple hydrological, hydrochemical, physicochemical and microbiological parameters, including discharge, turbidity, particle-size distributions, and E. coli, were measured with high temporal resolution during ten days of on-site monitoring. A complex contamination pattern due to anthropogenic and agricultural activities led to high E. coli concentrations (270 to >24,200 MPN/100ml) and a GLUC activity between 3.1 and 102.2 mMFU/100ml. A strong daily fluctuation pattern of GLUC activity and particle concentrations within small size classes (<10µm) could be observed, as demonstrated by autocorrelations. A Spearman's rank correlation analysis resulted in correlation coefficients of rs=0.56 for E. coli and GLUC activity and rs=0.54 for GLUC activity and the concentration of 2-3µm particles. On an event scale, correlations were found to be higher (rs=0.69 and 0.87, respectively). GLUC activity and E. coli displayed a general contamination pattern, but with significant differences in detail, which may be explained by interferences of e. g. viable but non-culturable cells. Although further evaluations are recommended, GLUC activity is a promising, complementary parameter for on-site and near real-time water quality monitoring.