Maciej Szewczyk

Yeast and human mitochondrial helicases.

Mitochondria are semiautonomous organelles which contain their own genome. Both maintenance and expression of mitochondrial DNA require activity of RNA and DNA helicases. In Saccharomyces cerevisiae the nuclear genome encodes four DExH/D superfamily members (MSS116, SUV3, MRH4, IRC3) that act as helicases and/or RNA chaperones. Their activity is necessary for mitochondrial RNA splicing, degradation, translation and genome maintenance. In humans the ortholog of SUV3 (hSUV3, SUPV3L1) so far is the best described mitochondrial RNA helicase. The enzyme, together with the matrix-localized pool of PNPase (PNPT1), forms an RNA-degrading complex called the mitochondrial degradosome, which localizes to distinct structures (D-foci). Global regulation of mitochondrially encoded genes can be achieved by changing mitochondrial DNA copy number. This way the proteins involved in its replication, like the Twinkle helicase (c10orf2), can indirectly regulate gene expression. Here, we describe yeast and human mitochondrial helicases that are directly involved in mitochondrial RNA metabolism, and present other helicases that participate in mitochondrial DNA replication and maintenance. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.

Upconverting/magnetic: Gd2O3:(Er3+,Yb3+,Zn2+) nanoparticles for biological applications: effect of Zn2+ doping

Upconverting Gd2O3 nanoparticles (NPs) doped 1% Er3+ and 18% Yb3+ permits one to perform optical imaging. Because of the presence of Gd3+ they are useful in MRI. The main challenge is to enhance the NPs upconversion efficiency. As a result of co-doping the NPs with Zn2+ ions, achieved using microwave-induced solution combustion synthesis, we obtained optimal upconversion quantum yields (UQYs). The breakdown of the local crystal field symmetry around the rare earth ions, maximal in the presence of 5% of zinc, may be responsible for the highest observed UQY. The upconversion of IR light results in emission of visible red light mainly at 660 nm and at 550 nm. Optimized red photoluminescence of the samples observed in an organic environment was examined as a function of the laser power density to explain the mechanism of the upconversion emission. Paramagnetic properties of the NPs were determined by superconducting quantum interference device measurements. The non-functionalized nanoparticles incubated with HeLa cells were endocytosed and imaged by confocal laser scanning microscopy. We investigated their localization inside HeLa cells for various incubation times and NPs concentrations. PrestoBlue toxicity assay was performed to test the NPs bio-efficacy.

Dedicated surveillance mechanism controls G-quadruplex forming non-coding RNAs in human mitochondria

The GC skew in vertebrate mitochondrial genomes results in synthesis of RNAs that are prone to form G-quadruplexes (G4s). Such RNAs, although mostly non-coding, are transcribed at high rates and are degraded by an unknown mechanism. Here we describe a dedicated mechanism of degradation of G4-containing RNAs, which is based on cooperation between mitochondrial degradosome and quasi-RNA recognition motif (qRRM) protein GRSF1. This cooperation prevents accumulation of G4-containing transcripts in human mitochondria. In vitro reconstitution experiments show that GRSF1 promotes G4 melting that facilitates degradosome-mediated decay. Among degradosome and GRSF1 regulated transcripts we identified one that undergoes post-transcriptional modification. We show that GRSF1 proteins form a distinct qRRM group found only in vertebrates. The appearance of GRSF1 coincided with changes in the mitochondrial genome, which allows the emergence of G4-containing RNAs. We propose that GRSF1 appearance is an evolutionary adaptation enabling control of G4 RNA.G-rich RNAs encoded in mitochondrial DNA are prone to form four-stranded structures called G-quadruplexes (G4s). Here the authors show using in vitro and in vivo approaches that GRSF1 promotes melting of G4 RNA structures in mtRNAs, thus leading to their decay by the hSuv3–PNPase complex.

Deficiencies in Natura 2000 for protecting recovering large carnivores: A spotlight on the wolf Canis lupus in Poland

If protected areas are to remain relevant in our dynamic world they must be adapted to changes in species ranges. In the EU one of the most notable such changes is the recent recovery of large carnivores, which are protected by Natura 2000 at the national and population levels. However, the Natura 2000 network was designed prior to their recent recovery, which raises the question whether the network is sufficient to protect the contemporary ranges of large carnivores. To investigate this question we evaluated Natura 2000 coverage of the three wolf Canis lupus populations in Poland. Wolf tracking data showed that wolves have recolonised almost all suitable habitat in Poland (as determined by a recent habitat suitability model), so we calculated the overlap between the Natura 2000 network and all wolf habitat in Poland. On the basis of published Natura 2000 criteria, we used 20% as the minimum required coverage. At the national level, wolves are sufficiently protected (22% coverage), but at the population level, the Baltic and Carpathian populations are far better protected (28 and 47%, respectively) than the endangered Central European Lowland population (12%). As Natura 2000 insufficiently protects the most endangered wolf population in Poland, we recommend expansion of Natura 2000 to protect at least an additional 8% of wolf habitat in western Poland, and discuss which specific forests are most in need of additional coverage. Implementation of these actions will have positive conservation implications and help Poland to fulfil its Habitats Directive obligations. As it is likely that similar gaps in Natura 2000 are arising in other EU member states experiencing large carnivore recoveries, particularly in Central Europe, we make the case for a flexible approach to Natura 2000 and suggest that such coverage evaluations may be beneficial elsewhere.

Human SUV3 helicase regulates growth rate of the HeLa cells and can localize in the nucleoli

The human SUV3 helicase (SUV3, hSUV3, SUPV3L1) is a DNA/RNA unwinding enzyme belonging to the class of DexH-box helicases. It localizes predominantly in the mitochondria, where it forms an RNA-degrading complex called mitochondrial degradosome with exonuclease PNP (polynucleotide phosphorylase). Association of this complex with the polyA polymerase can modulate mitochondrial polyA tails. Silencing of the SUV3 gene was shown to inhibit the cell cycle and to induce apoptosis in human cell lines. However, since small amounts of the SUV3 helicase were found in the cell nuclei, it was not clear whether the observed phenotypes of SUV3 depletion were of mitochondrial or nuclear origin. In order to answer this question we have designed gene constructs able to inhibit the SUV3 activity exclusively in the cell nuclei. The results indicate that the observed growth rate impairment upon SUV3 depletion is due to its nuclear function(s). Unexpectedly, overexpression of the nuclear-targeted wild-type copies of the SUV3 gene resulted in a higher growth rate. In addition, we demonstrate that the SUV3 helicase can be found in the HeLa cell nucleoli, but it is not detectable in the DNA-repair foci. Our results indicate that the nucleolar-associated human SUV3 protein is an important factor in regulation of the cell cycle.

Single-step synthesis of Er3+ and Yb3+ ions doped molybdate/Gd2O3 core–shell nanoparticles for biomedical imaging

Nanostructures as color-tunable luminescent markers have become major, promising tools for bioimaging and biosensing. In this paper separated molybdate/Gd2O3 doped rare earth ions (erbium, Er3+ and ytterbium, Yb3+) core-shell nanoparticles (NPs), were fabricated by a one-step homogeneous precipitation process. Emission properties were studied by cathodo- and photoluminescence. Scanning electron and transmission electron microscopes were used to visualize and determine the size and shape of the NPs. Spherical NPs were obtained. Their core-shell structures were confirmed by x-ray diffraction and energy-dispersive x-ray spectroscopy measurements. We postulated that the molybdate rich core is formed due to high segregation coefficient of the Mo ion during the precipitation. The calcination process resulted in crystallization of δ/ξ (core/shell) NP doped Er and Yb ions, where δ-gadolinium molybdates and ξ-molybdates or gadolinium oxide. We confirmed two different upconversion mechanisms. In the presence of molybdenum ions, in the core of the NPs, Yb3+-[Formula: see text] (∣2F7/2, 3T2〉) dimers were formed. As a result of a two 980 nm photon absorption by the dimer, we observed enhanced green luminescence in the upconversion process. However, for the shell formed by the Gd2O3:Er, Yb NPs (without the Mo ions), the typical energy transfer upconversion takes place, which results in red luminescence. We demonstrated that the NPs were transported into cytosol of the HeLa and astrocytes cells by endocytosis. The core-shell NPs are sensitive sensors for the environment prevailing inside (shorter luminescence decay) and outside (longer luminescence decay) of the tested cells. The toxicity of the NPs was examined using MTT assay.

Controlling the mitochondrial antisense – role of the SUV3-PNPase complex and its co-factor GRSF1 in mitochondrial RNA surveillance

ABSTRACT Transcription of the human mitochondrial genome produces a vast amount of non-coding antisense RNAs. These RNA species can form G-quadraplexes (G4), which affect their decay. We found that the mitochondrial degradosome, a complex of RNA helicase SUPV3L1 (best known as SUV3) and the ribonuclease PNPT1 (also known as PNPase), together with G4-melting protein GRSF1, is a key player in restricting antisense mtRNAs.

Upconversion fluorescence imaging of HeLa cells using ROS generating SiO2-coated lanthanide-doped NaYF4 nanoconstructs

Inorganic nanomaterials able to generate reactive oxygen species (ROS) are promising components for modern medical applications. Activated by near-infrared light, up-converting β-NaYF4 doped with Er3+–Yb3+ and Tm3+–Yb3+ pair ions nanoparticles (UCNPs), have a wide range of applications in biological imaging as compared to traditional reagents excited by ultra-violet or visible light. We analysed the green-red and the blue-red luminescence to explain the mechanism of the upconversion depended on the surface condition. The influence of SiO2 coating on the cytotoxicity of the as-produced UCNPs towards HeLa cancer cells was reported. We demonstrated a possibility of a direct UCNPs application to photodynamic therapy, without need to attach additional molecules to their surface. The presence of Tm3+–Yb3+ pair ions, thus ROS generation capability, renders the SiO2 shell coated nanoparticles to become potentially useful theranostic agent.