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Parkinson's disease (PD) is a common neurodegenerative disorder affecting mostly elderly people, although there is a group of patients developing so-called early-onset PD (EOPD). Mutations in the PARK2 gene are a common cause of autosomal recessive EOPD. PARK2 belongs to the family of extremely large human genes which are often localised in genomic common fragile sites (CFSs) and exhibit gross instability. PARK2 is located in the centre of FRA6E, the third most mutation-susceptible CFS of the human genome. The gene encompasses a region of 1.3 Mbp and, among its mutations, large rearrangements of single or multiple exons account for around 50%. We performed an analysis of the PARK2 gene in a group of 344 PD patients with EOPD and classical form of the disease. Copy number changes were first identified using multiplex ligation probe amplification (MLPA), with their ranges characterised by array comparative genomic hybridisation (aCGH). Exact breakpoints were mapped using direct sequencing. Rearrangements were found in eight subjects, including five deletions and three duplications. Rearrangements were mostly non-recurrent and no repetitive sequences or extended homologies were identified in the regions flanking breakpoint junctions. However, in most cases, 1-3 bp microhomologies were present, strongly suggesting that microhomology-mediated mechanisms, specifically non-homologous end joining (NHEJ) and fork stalling and template switching (FoSTeS)/microhomology-mediated break-induced replication (MMBIR), are predominantly involved in the rearrangement processes in this genomic region.

Human DIS3, the nuclear catalytic subunit of the exosome complex, contains exonucleolytic and endonucleolytic active domains. To identify DIS3 targets genome-wide, we combined comprehensive transcriptomic analyses of engineered HEK293 cells that expressed mutant DIS3, with Photoactivatable Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP) experiments. In cells expressing DIS3 with both catalytic sites mutated, RNAs originating from unannotated genomic regions increased ∼2.5-fold, covering ∼70% of the genome and allowing for thousands of novel transcripts to be discovered. Previously described pervasive transcription products, such as Promoter Upstream Transcripts (PROMPTs), accumulated robustly upon DIS3 dysfunction, representing a significant fraction of PAR-CLIP reads. We have also detected relatively long putative premature RNA polymerase II termination products of protein-coding genes whose levels in DIS3 mutant cells can exceed the mature mRNAs, indicating that production of such truncated RNA is a common phenomenon. In addition, we found DIS3 to be involved in controlling the formation of paraspeckles, nuclear bodies that are organized around NEAT1 lncRNA, whose short form was overexpressed in cells with mutated DIS3. Moreover, the DIS3 mutations resulted in misregulation of expression of ∼50% of transcribed protein-coding genes, probably as a secondary effect of accumulation of various noncoding RNA species. Finally, cells expressing mutant DIS3 accumulated snoRNA precursors, which correlated with a strong PAR-CLIP signal, indicating that DIS3 is the main snoRNA-processing enzyme. EXOSC10 (RRP6) instead controls the levels of the mature snoRNAs. Overall, we show that DIS3 has a major nucleoplasmic function in shaping the human RNA polymerase II transcriptome., (© 2015 Szczepińska et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Yeasts show remarkable variation in the organization of their mitochondrial genomes, yet there is little experimental data on organellar gene expression outside few model species. Candida albicans is interesting as a human pathogen, and as a representative of a clade that is distant from the model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Unlike them, it encodes seven Complex I subunits in its mtDNA. No experimental data regarding organellar expression were available prior to this study., Methods: We used high-throughput RNA sequencing and traditional RNA biology techniques to study the mitochondrial transcriptome of C. albicans strains BWP17 and SN148., Results: The 14 protein-coding genes, two ribosomal RNA genes, and 24 tRNA genes are expressed as eight primary polycistronic transcription units. We also found transcriptional activity in the noncoding regions, and antisense transcripts that could be a part of a regulatory mechanism. The promoter sequence is a variant of the nonanucleotide identified in other yeast mtDNAs, but some of the active promoters show significant departures from the consensus. The primary transcripts are processed by a tRNA punctuation mechanism into the monocistronic and bicistronic mature RNAs. The steady state levels of various mature transcripts exhibit large differences that are a result of posttranscriptional regulation. Transcriptome analysis allowed to precisely annotate the positions of introns in the RNL (2), COB (2) and COX1 (4) genes, as well as to refine the annotation of tRNAs and rRNAs. Comparative study of the mitochondrial genome organization in various Candida species indicates that they undergo shuffling in blocks usually containing 2-3 genes, and that their arrangement in primary transcripts is not conserved. tRNA genes with their associated promoters, as well as GC-rich sequence elements play an important role in these evolutionary events., Conclusions: The main evolutionary force shaping the mitochondrial genomes of yeasts is the frequent recombination, constantly breaking apart and joining genes into novel primary transcription units. The mitochondrial transcription units are constantly rearranged in evolution shaping the features of gene expression, such as the presence of secondary promoter sites that are inactive, or act as "booster" promoters, simplified transcriptional regulation and reliance on posttranscriptional mechanisms.

The present study explored the systematic inventory of Echinops L. (Asteraceae) of Saudi Arabia, with special reference to the molecular typing of Echinops abuzinadianus Chaudhary, an endemic species to Saudi Arabia, based on the internal transcribed spacer (ITS) sequences (ITS1-5.8S-ITS2) of nuclear ribosomal DNA. A sequence similarity search using BLAST and a phylogenetic analysis of the ITS sequence of E. abuzinadianus revealed a high level of sequence similarity with E. glaberrimus DC. (section Ritropsis). The novel primary sequence and the secondary structure of ITS2 of E. abuzinadianus could potentially be used for molecular genotyping.

In recent years, cancer stem cells (CSCs)/tumor initiating cells (TICs) have been identified inside different tumors. However, currently used anti-cancer therapies are mostly directed against somatic tumor cells without targeting CSCs/TICs. CSCs/TICs also gain resistance to chemotherapies/radiotherapies. For the development of efficient treatment strategies, choosing the best method for isolation and characterization of CSCs/TICs is still debated among the scientific community. In this review, we summarize recent data concerning isolation techniques for CSCs using magnetic cell sorting and flow cytometry. The review focuses on the strategies for sample preparation during flow cytometric analysis, elaborating biomarkers such as CXCR4, CD105, and CD133. In addition, functional properties characteristic of CSCs/TICs using side population selection through Hoechst 33342 dye, aldehyde dehydrogenase 1, dye-cycle violet, and rhodamine 123 are also discussed. We also include a special focus on enriching CSCs/TICs using three-dimensional cell culture models such as agarose-agarose microbeads and sphere formation.

Replication of the mitochondrial genome depends on the single DNA polymerase (pol gamma). Mutations in the POLG gene, encoding the catalytic subunit of the human polymerase gamma, have been linked to a wide variety of mitochondrial disorders that show remarkable heterogeneity, with more than 200 sequence variants, often very rare, found in patients. The pathogenicity and dominance status of many such mutations remain, however, unclear. Remarkable structural and functional conservation of human POLG and its S. cerevisiae ortholog (Mip1p) led to the development of many successful yeast models, enabling to study the phenotype of putative pathogenic mutations. In a group of patients with suspicion of mitochondrial pathology, we identified five novel POLG sequence variants, four of which (p.Arg869Ter, p.Gln968Glu, p.Thr1053Argfs*6, and p.Val1106Ala), together with one previously known but uncharacterised variant (p.Arg309Cys), were amenable to modelling in yeast. Familial analysis indicated causal relationship of these variants with disease, consistent with autosomal recessive inheritance. To investigate the effect of these sequence changes on mtDNA replication, we obtained the corresponding yeast mip1 alleles (Arg265Cys, Arg672Ter, Arg770Glu, Thr809Ter, and Val863Ala, respectively) and tested their effect on mitochondrial genome stability and replication fidelity. For three of them (Arg265Cys, Arg672Ter, and Thr809Ter), we observed a strong, partially dominant phenotype of a complete loss of functional mtDNA, whereas the remaining two led to partial mtDNA depletion and significant increase in point mutation frequencies. These results show good correlation with the severity of symptoms observed in patients and allow to establish these variants as pathogenic mutations.

Mitochondrial DNA was found to be highly mutated in colorectal cancer cells. One of the key molecules involved in the maintenance of the mitochondrial genome is the nuclear-encoded polymerase gamma. The aim of our study was to determine if there is a link between polymorphisms within the polymerase gamma gene (POLG) and somatic mutations within the mitochondrial genome in cancer cells. We investigated POLG sequence variability in 50 colorectal cancer patients whose complete mitochondrial genome sequences were determined. Relative mtDNA copy number was also determined. We identified 251 sequence variants in the POLG gene. Most of them were germline-specific (∼92%). Twenty-one somatic changes in POLG were found in 10 colorectal cancer patients. We have found no association between the occurrence of mtDNA somatic mutations and the somatically occurring variants in POLG. MtDNA content was reduced in patients carrying somatic variants in POLG or germline nucleotide variants located in the region encoding the POLG polymerase domain, but the difference did not reach statistical significance. Our findings suggest that somatic mtDNA mutations occurring in colorectal cancer are not a consequence of somatic mutations in POLG. Nevertheless, POLG nucleotide variants may lead to a decrease in mtDNA content, and consequently result in mitochondrial dysfunction., (© 2015 John Wiley & Sons Ltd/University College London.)

The biosynthesis of ribosomal RNA and its incorporation into functional ribosomes is an essential and intricate process that includes production of mature ribosomal RNA from large precursors. Here, we analyse the contribution of the plant exosome and its co-factors to processing and degradation of 18S pre-RNAs in Arabidopsis thaliana. Our data show that, unlike in yeast and humans, an RRP6 homologue, the nucleolar exoribonuclease RRP6L2, and the exosome complex, together with RRP44, function in two distinct steps of pre-18S rRNA processing or degradation in Arabidopsis. In addition, we identify TRL (TRF4/5-like) as the terminal nucleotidyltransferase that is mainly responsible for oligoadenylation of rRNA precursors in Arabidopsis. We show that TRL is required for efficient elimination of the excised 5' external transcribed spacer and of 18S maturation intermediates that escaped 5' processing. Our data also suggest involvement of additional nucleotidyltransferases, including terminal uridylyltransferase(s), in modifying rRNA processing intermediates in plants., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Non-consumptive effects (NCEs) of predators are part of the complex interactions among insect natural enemies and prey. NCEs have been shown to significantly affect prey foraging and feeding. Leafhopper's (Auchenorrhyncha) lengthy phloem feeding bouts may play a role in pathogen transmission in vector species and also exposes them to predation risk. However, NCEs on leafhoppers have been scarcely studied, and we lack basic information about how anti-predator behaviour influences foraging and feeding in these species. Here we report a study on non-consumptive and consumptive predator-prey interactions in a naturally co-occurring spider-leafhopper system. In mesocosm arenas we studied movement patterns during foraging and feeding of the leafhopper Psammotettix alienus in the presence of the spider predator Tibellus oblongus. Leafhoppers delayed feeding and fed much less often when the spider was present. Foraging movement pattern changed under predation risk: movements became more frequent and brief. There was considerable individual variation in foraging movement activity. Those individuals that increased movement activity in the presence of predators exposed themselves to higher predation risk. However, surviving individuals exhibited a 'cool headed' reaction to spider presence by moving less than leafhoppers in control trials. No leafhoppers were preyed upon while feeding. We consider delayed feeding as a "paradoxical" antipredator tactic, since it is not necessarily an optimal strategy against a sit-and-wait generalist predator.

Methanogenic Archaea produce approximately one billion tons of methane annually, but their biology remains largely unknown. This is partially due to the large phylogenetic and phenotypic diversity of this group of organisms, which inhabit various anoxic environments including peatlands, freshwater sediments, landfills, anaerobic digesters and the intestinal tracts of ruminants. Research is also hampered by the inability to cultivate methanogenic Archaea. Therefore, biodiversity studies have relied on the use of 16S rRNA and mcrA [encoding the α subunit of the methyl coenzyme M (methyl-CoM) reductase] genes as molecular markers for the detection and phylogenetic analysis of methanogens. Here, we describe four novel molecular markers that should prove useful in the detailed analysis of methanogenic consortia, with a special focus on methylotrophic methanogens. We have developed and validated sets of degenerate PCR primers for the amplification of genes encoding key enzymes involved in methanogenesis: mcrB and mcrG (encoding β and γ subunits of the methyl-CoM reductase, involved in the conversion of methyl-CoM to methane), mtaB (encoding methanol-5-hydroxybenzimidazolylcobamide Co-methyltransferase, catalyzing the conversion of methanol to methyl-CoM) and mtbA (encoding methylated [methylamine-specific corrinoid protein]:coenzyme M methyltransferase, involved in the conversion of mono-, di- and trimethylamine into methyl-CoM). The sensitivity of these primers was verified by high-throughput sequencing of PCR products amplified from DNA isolated from microorganisms present in anaerobic digesters. The selectivity of the markers was analyzed using phylogenetic methods. Our results indicate that the selected markers and the PCR primer sets can be used as specific tools for in-depth diversity analyses of methanogenic consortia.

AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Diverse subcellular localizations of the huntingtin-containing inclusion bodies are frequently suspected of reflecting crucial divisions between different cellular pathways contributing to the pathophysiology of Huntington's disease. Here, we use a panel of different N-terminal huntingtin fragments overexpressed in transfected neuronal and non-neuronal cells to demonstrate that it is the length of the N-terminal huntingtin fragments rather than a presence of any specific amino acid sequences that determines the ratio between the nuclear and cytoplasmic inclusion bodies. Importantly, the length of those fragments does also seem to strongly influence the folding of the aggregating huntingtin species, as indicated by the apparent differences in their accessibility for different antibodies directed against particular subdomains within the N-terminal part of huntingtin, although these differences do not correlate with the peptides' ability to efficiently aggregate within the cell nucleus. Furthermore, the relatively long huntingtin fragment containing 588 amino acids of the reference sequence shows intracellular behavior that is substantially different from that exhibited by its shorter counterparts (containing either 171, 120, 89 or 64 amino acids), as this rarely aggregating peptide is not only accumulating in cytoplasmic inclusions of slightly different morphology but is also most strongly affected by the FLAG-tagging procedure that unexpectedly induces (or enhances) autophagy-related processes. Together, our results reveal a significant heterogeneity of the huntingtin accumulation patterns that are observed at the cellular level. These patterns are not only strongly dependent on both the length and the amino acid composition of the N-terminal huntingtin peptides but also seem to engage different cellular mechanisms implicated in the pathogenesis of Huntington's disease, including the non-proteasomal degradation of potentially toxic huntingtin forms., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Little is known about the RNA polymerase III (Pol III) complex assembly and its transport to the nucleus. We demonstrate that a missense cold-sensitive mutation, rpc128-1007, in the sequence encoding the C-terminal part of the second largest Pol III subunit, C128, affects the assembly and stability of the enzyme. The cellular levels and nuclear concentration of selected Pol III subunits were decreased in rpc128-1007 cells, and the association between Pol III subunits as evaluated by coimmunoprecipitation was also reduced. To identify the proteins involved in Pol III assembly, we performed a genetic screen for suppressors of the rpc128-1007 mutation and selected the Rbs1 gene, whose overexpression enhanced de novo tRNA transcription in rpc128-1007 cells, which correlated with increased stability, nuclear concentration, and interaction of Pol III subunits. The rpc128-1007 rbs1Δ double mutant shows a synthetic growth defect, indicating that rpc128-1007 and rbs1Δ function in parallel ways to negatively regulate Pol III assembly. Rbs1 physically interacts with a subset of Pol III subunits, AC19, AC40, and ABC27/Rpb5. Additionally, Rbs1 interacts with the Crm1 exportin and shuttles between the cytoplasm and nucleus. We postulate that Rbs1 binds to the Pol III complex or subcomplex and facilitates its translocation to the nucleus., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Leber hereditary optic neuropathy (LHON) is a genetic, maternally inherited disease caused by point mutations in the mitochondrial genome. LHON patients present with sudden, painless and usually bilateral loss of vision caused by optic nerve atrophy. The first clinical description of the disease was made by Theodor Leber, a German ophthalmologist, in 1871. Here we present his thorough notes about members of four families and their pedigrees. We also provide insights into the current knowledge about LHON pathology, genetics and treatment in comparison with Leber's findings., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Objective: The aim of this study was to analyze the intra-/interfamilial phenotypic heterogeneity due to variants at the highly evolutionary conservative p.Arg1596 residue in the Nav1.1 subunit., Materials/participants: Among patients referred for analysis of the SCN1A gene one recurrent, heritable mutation was found in families enrolled into the study. Probands from those families even clinically diagnosed with atypical Dravet syndrome (DS), generalized epilepsy with febrile seizures plus (GEFS+), and focal epilepsy, had heterozygous p.Arg1596 His/Cys missense substitutions, c.4787G>T and c.4786C>T in the SCN1A gene., Method: Full clinical evaluation, including cognitive development, neurological examination, EEGs, MRI was performed in probands and affected family members in developmental age. The whole SCN1A gene sequencing was performed for all probands. The exon 25, where the identified missense substitutions are localized, was directly analyzed for the other family members., Results: Mutation of the SCN1A p.1596Arg was identified in three families, in one case substitution p.Arg1596Cys and in two cases p.Arg1596His. Both mutations were previously described as pathogenic and causative for DS, GEFS+ and focal epilepsy. Spectrum of phenotypes among presented families with p.Arg1596 mutations shows heterogeneity ranged from asymptomatic cases, through FS and FS+ to GEFS+/Panayiotopoulos syndrome and epilepsies with and without febrile seizures, and epileptic encephalopathy such as DS. Phenotypes differ among patients displaying both focal and generalized epilepsies. Some patients demonstrated additionally Asperger syndrome and ataxia., Conclusion: Clinical picture heterogeneity of the patients carrying mutation of the same residue indicates the involvement of the other factors influencing the SCN1A gene mutations' penetrance., (Copyright © 2015 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Production of ribosomes relies on more than 200 accessory factors to ensure the proper sequence of steps and faultless assembly of ribonucleoprotein machinery. Among trans-acting factors are numerous enzymes, including ribonucleases responsible for processing the large rRNA precursor synthesized by RNA polymerase I that encompasses sequences corresponding to mature 18S, 5.8S, and 25/28S rRNA. In humans, the identity of most enzymes responsible for individual processing steps, including endoribonucleases that cleave pre-rRNA at specific sites within regions flanking and separating mature rRNA, remains largely unknown. Here, we investigated the role of hUTP24 in rRNA maturation in human cells. hUTP24 is a human homolog of the Saccharomyces cerevisiae putative PIN domain-containing endoribonuclease Utp24 (yUtp24), which was suggested to participate in the U3 snoRNA-dependent processing of yeast pre-rRNA at sites A0, A1, and A2. We demonstrate that hUTP24 interacts to some extent with proteins homologous to the components of the yeast small subunit (SSU) processome. Moreover, mutation in the putative catalytic site of hUTP24 results in slowed growth of cells and reduced metabolic activity. These effects are associated with a defect in biogenesis of the 40S ribosomal subunit, which results from decreased amounts of 18S rRNA as a consequence of inaccurate pre-rRNA processing at the 5'-end of the 18S rRNA segment (site A1). Interestingly, and in contrast to yeast, site A0 located upstream of A1 is efficiently processed upon UTP24 dysfunction. Finally, hUTP24 inactivation leads to aberrant processing of 18S rRNA 2 nucleotides downstream of the normal A1 cleavage site.

The present article highlights the diverse role of stem cells in normal kidney and renal cancer, with special emphasis on surface markers. Proteins such as CD105 and CD133 have been reported as being significant in clear cell renal cell carcinoma (ccRCC) cancer stem cells. The role of normal kidney progenitor cells and their surface markers is compared with the role of those surface markers in ccRCC. Subsequently, we state the current hypothesis about origin of tumour-initiating cells along with their clinical and prognostic potential in RCC. Finally, we present future perspectives with respect to recent studies.

The ethiology of colon cancer is largely dependent on inflammation driven oxidative stress. The analysis of 8-oxodeoxyguanosine (8-oxodGuo) level in leukocyte DNA of healthy controls (138 individuals), patients with benign adenomas (AD, 137 individuals) and with malignant carcinomas (CRC, 169 individuals) revealed a significant increase in the level of 8-oxodGuo in leukocyte DNA of AD and CRC patients in comparison to controls. The counteracting mechanism is base excision repair, in which OGG1 and PARP-1 play a key role. We investigated the level of PARP-1 and OGG1 mRNA and protein in diseased and marginal, normal tissues taken from AD and CRC patients and in leukocytes taken from the patients as well as from healthy subjects. In colon tumors the PARP-1 mRNA level was higher than in unaffected colon tissue and in polyp tissues. A high positive correlation was found between PARP-1 and OGG1 mRNA levels in all investigated tissues. This suggests reciprocal influence of PARP-1 and OGG1 on their expression and stability, and may contribute to progression of colon cancer. PARP-1 and OGG1 proteins level was several fold higher in polyps and CRC in comparison to normal colon tissues. Individuals bearing the Cys326Cys genotype of OGG1 were characterized by higher PARP-1 protein level in diseased tissues than the Ser326Cys and Ser326Ser genotypes. Aforementioned result may suggest that the diseased cells with polymorphic OGG1 recruit more PARP protein, which is necessary to remove 8-oxodGuo. Thus, patients with decreased activity of OGG1/polymorphism of the OGG1 gene and higher 8-oxodGuo level may be more susceptible to treatment with PARP-1 inhibitors.

The conservation of large carnivores is a formidable challenge for biodiversity conservation. Using a data set on the past and current status of brown bears (Ursus arctos), Eurasian lynx (Lynx lynx), gray wolves (Canis lupus), and wolverines (Gulo gulo) in European countries, we show that roughly one-third of mainland Europe hosts at least one large carnivore species, with stable or increasing abundance in most cases in 21st-century records. The reasons for this overall conservation success include protective legislation, supportive public opinion, and a variety of practices making coexistence between large carnivores and people possible. The European situation reveals that large carnivores and people can share the same landscape., (Copyright © 2014, American Association for the Advancement of Science.)

MGME1, also known as Ddk1 or C20orf72, is a mitochondrial exonuclease found to be involved in the processing of mitochondrial DNA (mtDNA) during replication. Here, we present detailed insights on the role of MGME1 in mtDNA maintenance. Upon loss of MGME1, elongated 7S DNA species accumulate owing to incomplete processing of 5' ends. Moreover, an 11-kb linear mtDNA fragment spanning the entire major arc of the mitochondrial genome is generated. In contrast to control cells, where linear mtDNA molecules are detectable only after nuclease S1 treatment, the 11-kb fragment persists in MGME1-deficient cells. In parallel, we observed characteristic mtDNA duplications in the absence of MGME1. The fact that the breakpoints of these mtDNA rearrangements do not correspond to either classical deletions or the ends of the linear 11-kb fragment points to a role of MGME1 in processing mtDNA ends, possibly enabling their repair by homologous recombination. In agreement with its functional involvement in mtDNA maintenance, we show that MGME1 interacts with the mitochondrial replicase PolgA, suggesting that it is a constituent of the mitochondrial replisome, to which it provides an additional exonuclease activity. Thus, our results support the viewpoint that MGME1-mediated mtDNA processing is essential for faithful mitochondrial genome replication and might be required for intramolecular recombination of mtDNA., (© The Author 2014. Published by Oxford University Press.)

Perfringolysin O (PFO) is a toxic protein that binds to cholesterol-containing membranes, oligomerizes, and forms a β-barrel transmembrane pore, leading to cell lysis. Previous studies have uncovered the sequence of events in this multistage structural transition to a considerable detail, but the underlying molecular mechanisms are not yet fully understood. By measuring hydrogen-deuterium exchange patterns of peptide bond amide protons monitored by mass spectrometry (MS), we have mapped structural changes in PFO and its variant bearing a point mutation during incorporation to the lipid environment. We have defined all regions that undergo structural changes caused by the interaction with the lipid environment both in wild-type PFO, thus providing new experimental constraints for molecular modeling of the pore formation process, and in a point mutant, W165T, for which the pore formation process is known to be inefficient. We have demonstrated that point mutation W165T causes destabilization of protein solution structure, strongest for domain D1, which interrupts the pathway of structural transitions in other domains necessary for proper oligomerization in the membrane. In PFO, the strongest changes accompanying binding to the membrane focus in D1; the C-terminal part of D4; and strands β1, β4, and β5 of D3. These changes were much weaker for PFO(W165T) lipo where substantial stabilization was observed only in D4 domain. In this study, the application of hydrogen-deuterium exchange analysis monitored by MS provided new insight into conformational changes of PFO associated with the membrane binding, oligomerization, and lytic pore formation., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Oxidative-stress-driven lipid peroxidation (LPO) is involved in the pathogenesis of several human diseases, including cancer. LPO products react with cellular proteins changing their properties, and with DNA bases to form mutagenic etheno-DNA adducts, removed from DNA mainly by the base excision repair (BER) pathway. One of the major reactive aldehydes generated by LPO is 4-hydroxy-2-nonenal (HNE). We investigated the effect of HNE on BER enzymes in human cells and in vitro. K21 cells pretreated with physiological HNE concentrations were more sensitive to oxidative and alkylating agents, H2O2 and MMS, than were untreated cells. Detailed examination of the effects of HNE on particular stages of BER in K21 cells revealed that HNE decreases the rate of excision of 1,N(6)-ethenoadenine (ɛA) and 3,N(4)-ethenocytosine (ɛC), but not of 8-oxoguanine. Simultaneously HNE increased the rate of AP-site incision and blocked the re-ligation step after the gap-filling by DNA polymerases. This suggested that HNE increases the number of unrepaired single-strand breaks (SSBs) in cells treated with oxidizing or methylating agents. Indeed, preincubation of cells with HNE and their subsequent treatment with H2O2 or MMS increased the number of nuclear poly(ADP-ribose) foci, known to appear in cells in response to SSBs. However, when purified BER enzymes were exposed to HNE, only ANPG and TDG glycosylases excising ɛA and ɛC from DNA were inhibited, and only at high HNE concentrations. APE1 endonuclease and 8-oxoG-DNA glycosylase 1 (OGG1) were not inhibited. These results indicate that LPO products exert their promutagenic action not only by forming DNA adducts, but in part also by compromising the BER pathway., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Variation in the intracellular percentage of normal and mutant mitochondrial DNAs (mtDNA) (heteroplasmy) can be associated with phenotypic heterogeneity in mtDNA diseases. Individuals that inherit the common disease-causing mtDNA tRNA(Leu(UUR)) 3243A>G mutation and harbor ∼10-30% 3243G mutant mtDNAs manifest diabetes and occasionally autism; individuals with ∼50-90% mutant mtDNAs manifest encephalomyopathies; and individuals with ∼90-100% mutant mtDNAs face perinatal lethality. To determine the basis of these abrupt phenotypic changes, we generated somatic cell cybrids harboring increasing levels of the 3243G mutant and analyzed the associated cellular phenotypes and nuclear DNA (nDNA) and mtDNA transcriptional profiles by RNA sequencing. Small increases in mutant mtDNAs caused relatively modest defects in oxidative capacity but resulted in sharp transitions in cellular phenotype and gene expression. Cybrids harboring 20-30% 3243G mtDNAs had reduced mtDNA mRNA levels, rounded mitochondria, and small cell size. Cybrids with 50-90% 3243G mtDNAs manifest induction of glycolytic genes, mitochondrial elongation, increased mtDNA mRNA levels, and alterations in expression of signal transduction, epigenomic regulatory, and neurodegenerative disease-associated genes. Finally, cybrids with 100% 3243G experienced reduced mtDNA transcripts, rounded mitochondria, and concomitant changes in nuclear gene expression. Thus, striking phase changes occurred in nDNA and mtDNA gene expression in response to the modest changes of the mtDNA 3243G mutant levels. Hence, a major factor in the phenotypic variation in heteroplasmic mtDNA mutations is the limited number of states that the nucleus can acquire in response to progressive changes in mitochondrial retrograde signaling.

Retrotransposons (RTs) are major components of most eukaryotic genomes. They are ubiquitous, dispersed throughout the genome, and their abundance correlates with genome size. Their copy-and-paste lifestyle in the genome consists of three molecular steps involving transcription of an RNA copy from the genomic RT, followed by reverse transcription to generate cDNA, and finally, reintegration into a new location in the genome. This process leads to new genomic insertions without excision of the original element. The target sites of insertions are relatively random and independent for different taxa; however, some elements cluster together in 'repeat seas' or have a tendency to cluster around the centromeres and telomeres. The structure and copy number of retrotransposon families are strongly influenced by the evolutionary history of the host genome. Molecular markers play an essential role in all aspects of genetics and genomics, and RTs represent a powerful tool compared with other molecular and morphological markers. All features of integration activity, persistence, dispersion, conserved structure and sequence motifs, and high copy number suggest that RTs are appropriate genomic features for building molecular marker systems. To detect polymorphisms for RTs, marker systems generally rely on the amplification of sequences between the ends of the RT, such as (long-terminal repeat)-retrotransposons and the flanking genomic DNA. Here, we review the utility of some commonly used PCR retrotransposon-based molecular markers, including inter-primer binding sequence (IPBS), sequence-specific amplified polymorphism (SSAP), retrotransposon-based insertion polymorphism (RBIP), inter retrotransposon amplified polymorphism (IRAP), and retrotransposon-microsatellite amplified polymorphism (REMAP).

Oligomers formed by amyloid β (Aβ) peptide are widely believed to be the main neurotoxic agent in Alzheimer's disease. Studies discovered a broad variety of oligomeric forms, which display different levels of toxicity. Some of these forms may further assemble into mature fibrils, while other might be off-pathway from conversion to fibrils and assemble into alternative forms. To better understand a relationship between the structure and toxicity of Aβ oligomers, we require systematic characterization and classification of all possible forms, facilitating rational design of the beneficial modifiers of their activity. In previous ion mobility analysis of Aβ1-40 oligomers, we have detected the coexistence of two alternative structural forms (compact and extended) in a pool of low-order Aβ1-40 oligomers. These forms may represent two pathways of the oligomer evolution, leading either to fibrils or to off-pathway oligomers, which are potential candidates for the neurotoxic species. Here, we have analyzed the impact of incubation time, the presence of selected metal ions and the effect of a series of point mutations on mutual population of alternative forms. We have shown that a salt bridge D23K28 provides stabilization of the compact form whereas G25 is required for the existence of the extended form. We have found that binding of metal ions also stabilizes the compact form. These results improve our understanding of the possible molecular mechanism of the bifurcation of structural evolution of non-monomeric Aβ species into an off-fibril pathway, ultimately leading to the formation of potentially neurotoxic species., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The discipline taxonomy (the science of naming and classifying organisms, the original bioinformatics and a basis for all biology) is fundamentally important in ensuring the quality of life of future human generation on the earth; yet over the past few decades, the teaching and research funding in taxonomy have declined because of its classical way of practice which lead the discipline many a times to a subject of opinion, and this ultimately gave birth to several problems and challenges, and therefore the taxonomist became an endangered race in the era of genomics. Now taxonomy suddenly became fashionable again due to revolutionary approaches in taxonomy called DNA barcoding (a novel technology to provide rapid, accurate, and automated species identifications using short orthologous DNA sequences). In DNA barcoding, complete data set can be obtained from a single specimen irrespective to morphological or life stage characters. The core idea of DNA barcoding is based on the fact that the highly conserved stretches of DNA, either coding or non coding regions, vary at very minor degree during the evolution within the species. Sequences suggested to be useful in DNA barcoding include cytoplasmic mitochondrial DNA (e.g. cox1) and chloroplast DNA (e.g. rbcL, trnL-F, matK, ndhF, and atpB rbcL), and nuclear DNA (ITS, and house keeping genes e.g. gapdh). The plant DNA barcoding is now transitioning the epitome of species identification; and thus, ultimately helping in the molecularization of taxonomy, a need of the hour. The 'DNA barcodes' show promise in providing a practical, standardized, species-level identification tool that can be used for biodiversity assessment, life history and ecological studies, forensic analysis, and many more.

Over 90% of Leber's hereditary optic neuropathy (LHON) is caused by one of three mtDNA mutations (m.11778A>G, m.3460G>A, m.14484T>C). The remaining cases are due to rare mutations in different genes encoding subunits of the respiratory chain. The proband is a 17-year-old male with symptoms of optic nerve atrophy. No common LHON mutations were found, but detailed sequencing identified a rare, homoplasmic mutation m.3635G>A in the ND1 gene., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Oligomeric forms of Aβ peptide are most likely the main synaptotoxic and neurotoxic agent in Alzheimer's disease. Toxicity of various Aβ oligomeric forms has been confirmed in vivo and also in vitro. However, in vitro preparations were found to be orders of magnitude less toxic than oligomers obtained from in vivo sources. This difference can be explained by the presence of a covalent cross-link, which would stabilize the oligomer. In the present work, we have characterized the structural properties of Aβ dimers and trimers stabilized by di- and tri-tyrosine cross-links. Using ion mobility mass spectrometry we have compared the collisional cross-section of non-cross-linked and cross-linked species. We have found that the presence of cross-links does not generate new unique forms but rather shifts the equilibrium towards more compact oligomer types that can also be detected for non-cross-linked peptide. In consequence, more extended forms, probable precursors of off-pathway oligomeric species, become relatively destabilized in cross-linked oligomers and the pathway of oligomer evolution becomes redirected towards fibrillar structures.

The ALKBH family of Fe(II) and 2-oxoglutarate dependent oxygenases comprises enzymes that display sequence homology to AlkB from E. coli, a DNA repair enzyme that uses an oxidative mechanism to dealkylate methyl and etheno adducts on the nucleobases. Humans have nine different ALKBH proteins, ALKBH1-8 and FTO. Mammalian and plant ALKBH8 are tRNA hydroxylases targeting 5-methoxycarbonylmethyl-modified uridine (mcm5U) at the wobble position of tRNAGly(UCC). In contrast, the genomes of some bacteria encode a protein with strong sequence homology to ALKBH8, and robust DNA repair activity was previously demonstrated for one such protein. To further explore this apparent functional duality of the ALKBH8 proteins, we have here enzymatically characterized a panel of such proteins, originating from bacteria, protozoa and mimivirus. All the enzymes showed DNA repair activity in vitro, but, interestingly, two protozoan ALKBH8s also catalyzed wobble uridine modification of tRNA, thus displaying a dual in vitro activity. Also, we found the modification status of tRNAGly(UCC) to be unaltered in an ALKBH8 deficient mutant of Agrobacterium tumefaciens, indicating that bacterial ALKBH8s have a function different from that of their eukaryotic counterparts. The present study provides new insights on the function and evolution of the ALKBH8 family of proteins.

Oxidative stress via redox reactions can regulate DNA repair pathways. The base excision repair (BER) enzyme apurinic/apyrimidinic endonuclease 1 (APE1) is a key player in the redox regulation of DNA repair. Environmental factors can alter the methylation of DNA repair genes, change their expression and thus modulate BER activity and susceptibility to oxidative DNA damage. Therefore, we hypothesized that epigenetic modifications play a role in the redox regulation of APE1 in hippocampi of newborns and investigated the effect of supplementation of pregnant sows with a diet enriched in antioxidants and other nutrients on oxidative stress, DNA methylation and DNA repair in their offspring. High levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and low levels of glutathione were detected in control piglets after birth compared with supplemented piglets, indicating the presence of oxidative stress. In control animals, this oxidative stress was associated with genomic DNA demethylation, decreased APE1 promoter methylation, increased APE1 expression and with slightly but not statistically significant increased BER-related DNA incision activity. Supplementation of piglets with antioxidants and other nutrients significantly lowered 8-oxodG levels compared to control animals, which was accompanied by overall lower APE1 promoter methylation and enhanced APE1 expression at day 7-28 after birth in supplemented piglets, although DNA incision activity was not significantly different between groups. Preliminary attempts to study the interaction between redox and epigenetic regulatory mechanisms revealed an inverse correlation between APE1 expression and methylation of CpG-sites 11 and 13 in the promoter region, which according to Genomatix "MatInspector" are located in the core binding sites of redox-sensitive transcription factors. We are the first to study methylation of the APE1 promoter and its role in mediating the functional effects of redox reactions induced by oxidative stress. Epigenetic and redox mechanisms may interact in regulating APE1-related DNA repair processes, involving redox-sensitive TFs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Genomic retrotransposons (RTs) are major components of most plant genomes. They spread throughout the genomes by a process termed retrotransposition, which consists of reverse transcription and reinsertion of the copied element into a new genomic location (a copy-and-paste system). Abiotic and biotic stresses activate long-terminal repeat (LTR) RTs in photosynthetic eukaryotes from algae to angiosperms. LTR RTs could represent a threat to the integrity of host genomes because of their activity and mutagenic potential by epigenetic regulation. Host genomes have developed mechanisms to control the activity of the retroelements and their mutagenic potential. Some LTR RTs escape these defense mechanisms, and maintain their ability to be activated and transpose as a result of biotic or abiotic stress stimuli. These stimuli include pathogen infection, mechanical damage, in vitro tissue culturing, heat, drought and salt stress, generation of doubled haploids, X-ray irradiation and many others. Reactivation of LTR RTs differs between different plant genomes. The expression levels of reactivated RTs are influenced by the transcriptional and post-transcriptional gene silencing mechanisms (e.g. DNA methylation, heterochromatin formation and RNA interference). Moreover, the insertion of RTs (e.g. Triticum aestivum L. Wis2-1A) into or next to coding regions of the host genome can generate changes in the expression of adjacent host genes of the host. In this paper, we review the ways that plant genomic LTR RTs are activated by environmental stimuli to affect restructuring and diversification of the host genome.

Background: Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized., Methodology/principal Findings: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+) and Ca(2+) (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+), Co(2+) and Fe(2+) cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6-7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 μM(-1) · min(-1), respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1., Conclusions/significance: Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.

We present a general probabilistic framework for predicting the substrate specificity of enzymes. We designed this approach to be easily applicable to different organisms and enzymes. Therefore, our predictive models do not rely on species-specific properties and use mostly sequence-derived data. Maximum Likelihood optimization is used to fine-tune model parameters and the Akaike Information Criterion is employed to overcome the issue of correlated variables. As a proof-of-principle, we apply our approach to predicting general substrate specificity of yeast methyltransferases (MTases). As input, we use several physico-chemical and biological properties of MTases: structural fold, isoelectric point, expression pattern and cellular localization. Our method accurately predicts whether a yeast MTase methylates a protein, RNA or another molecule. Among our experimentally tested predictions, 89% were confirmed, including the surprising prediction that YOR021C is the first known MTase with a SPOUT fold that methylates a substrate other than RNA (protein). Our approach not only allows for highly accurate prediction of functional specificity of MTases, but also provides insight into general rules governing MTase substrate specificity.

Cancer is a major health problem and cause of death worldwide that accounted for 7.6 million deaths in 2008, which is projected to continue rising with an estimated 13.1 million deaths in 2030 according to WHO. Breast cancer is the leading cause of cancer-based death among women around the world and its incidence is increasing annually with a similar tendency. In contrast, renal cell carcinoma accounts for only 3% of total human malignancies but it is still the most common type of urological cancer with a high prevalence in elderly men (>60 years of age). There are several factors linked with the development of renal cell cancer only, while others are connected only with breast cancer. Genetic risk factors and smoking are the factors which contribute to carcinogenesis in general. Some evidence exists indicating that vitamin D receptor (VDR) gene polymorphisms are associated with both breast and renal cancer; therefore, we put forward the hypothesis that polymorphisms in the VDR gene may influence both the occurrence risks of these cancers and their prognosis. However, the relationship between VDR polymorphisms and these two specific cancers remains a controversial hypothesis, and consequently needs further confirmation via clinical research together with genetic investigations. Here, we aimed to assess the correlation between the different alleles of VDR gene polymorphisms and renal cell cancer and breast cancer risks separately through a systematic review of the present literature. In contrast, this analysis has revealed that some VDR gene polymorphisms, such as: Bsm1, poly(A), Taq1, Apa1, are to some extent associated with breast cancer risk. Other polymorphisms were found to be significantly associated with renal cell cancer. Namely, they were Fok1, Bsm1, Taq1 and Apa1, which encode proteins participating mainly in proliferation, apoptosis and cell cycle regulation. However, data concerning renal cancer are not sufficient to firmly establish the VDR gene polymorphism association.

Twenty-seven grapevine ( Vitis vinifera L. ) varieties within 12 putative berry colour variation groups ( conculta ) were genotyped with 14 highly polymorphic microsatellite (simple sequence repeats (SSR)) markers. Three additional oligonucleotide primers were applied for the detection of the Gret1 retroelement insertion in the promoter region of VvMybA1 transcription factor gene regulating the UFGT (UDP-glucose: flavonoid 3-O-glucosyltransferase) activity. UFGT is the key enzyme of the anthocyanin biosynthetic pathway. SSR results proved that the analysed cultivars can be grouped only into nine concultas , the other three putative berry colour variant groups consist of homonyms as a consequence of misnaming. In the case of Sárfehér-Sárpiros, Delaware red-Delaware white and Járdovány fekete-Járdovány fehér, it was attested that they are not bud sports, but homonyms. Some conculta members could be differentiated according to the presence or the absence of the Gret1 retroelement (Chasselas, Furmint and Lisztes), while others, Bajor, Bakator, Gohér and Traminer conculta members, remained indistinguishable either by the microsatellites or the Gret1 -based method.

Mitochondrial diseases may cause a wide range of central and peripheral nervous system disorders, as well as muscle disorders. The diagnostic workup routinely includes electrophysiological, morphological, neuroimaging and genetic studies. In some cases, the diagnosis may be ascertained only when mitochondrial DNA (mtDNA) examination in the muscle is performed. We report on a case of a 24-year-old woman, with a 7-year history of slowly progressive cerebellar syndrome and bilateral ptosis. Mitochondrial encephalomyopathy was suspected, based on the clinical picture and results of examinations, but the typical red ragged fibers were not found in the muscle biopsy. The results of molecular analysis of mtDNA showed a mtDNA deletion in the muscle and, on a level detectable only with polymerase chain reaction method, in blood leukocytes. This case emphasizes the important role of mtDNA studies in muscle in nonspecific multisystem mitochondrial disorders, even without clinical muscle involvement., (Copyright © 2014 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

The introduction of anti-angiogenic drugs especially tyrosine kinase inhibitors (TKIs) was a breakthrough in the treatment of renal cell carcinoma (RCC). Although TKIs have significantly improved outcome in patients with metastatic disease, the majority still develop resistance over time. Because different combinations and sequences of TKIs are tested in clinical trials, resistance patterns and mechanisms underlying this phenomenon should be thoroughly investigated. From a clinical point of view, resistance occurs either as a primary phenomenon (intrinsic) or as a secondary phenomenon related to various escape/evasive mechanisms that the tumor develops in response to vascular endothelial growth factor (VEGF) inhibition. Intrinsic resistance is less common, and related to the primary redundancy of available angiogenic signals from the tumor, causing unresponsiveness to VEGF-targeted therapies. Acquired resistance in tumors is associated with activation of an angiogenic switch which leads to either upregulation of the existing VEGF pathway or recruitment of alternative factors responsible for tumor revascularization. Multiple mechanisms can be involved in different tumor settings that contribute both to evasive and intrinsic resistance, and current endeavor aims to identify these processes and assess their importance in clinical settings and design of pharmacological strategies that lead to enduring anti-angiogenic therapies., (© 2013. Published by Elsevier B.V. All rights reserved.)

Determination of RNA stability is one of the basic issues addressed in studies of RNA metabolism. In a standard approach used for RNA half-life measurement synthesis of RNA is inhibited and then the steady-state level of RNA is quantified and used for calculations. Here, we present an optimized protocol for mitochondrial RNA stability studies without perturbation of transcription and present results produced for the mitochondrial CytB messenger RNA. This method was originally described for nuclear transcripts and involves metabolic labeling of RNA with 4-thiouridine.

4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

DNA glycosylases from the Fpg/Nei structural superfamily are base excision repair enzymes involved in the removal of a wide variety of mutagen and potentially lethal oxidized purines and pyrimidines. Although involved in genome stability, the recent discovery of synthetic lethal relationships between DNA glycosylases and other pathways highlights the potential of DNA glycosylase inhibitors for future medicinal chemistry development in cancer therapy. By combining biochemical and structural approaches, the physical target of 2-thioxanthine (2TX), an uncompetitive inhibitor of Fpg, was identified. 2TX interacts with the zinc finger (ZnF) DNA binding domain of the enzyme. This explains why the zincless hNEIL1 enzyme is resistant to 2TX. Crystal structures of the enzyme bound to DNA in the presence of 2TX demonstrate that the inhibitor chemically reacts with cysteine thiolates of ZnF and induces the loss of zinc. The molecular mechanism by which 2TX inhibits Fpg may be generalized to all prokaryote and eukaryote ZnF-containing Fpg/Nei-DNA glycosylases. Cell experiments show that 2TX can operate in cellulo on the human Fpg/Nei DNA glycosylases. The atomic elucidation of the determinants for the interaction of 2TX to Fpg provides the foundation for the future design and synthesis of new inhibitors with high efficiency and selectivity., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

hDIS3 is a mainly nuclear, catalytic subunit of the human exosome complex, containing exonucleolytic (RNB) and endonucleolytic (PIN) active domains. Mutations in hDIS3 have been found in ∼10% of patients with multiple myeloma (MM). Here, we show that these mutations interfere with hDIS3 exonucleolytic activity. Yeast harboring corresponding mutations in DIS3 show growth inhibition and changes in nuclear RNA metabolism typical for exosome dysfunction. Construction of a conditional DIS3 knockout in the chicken DT40 cell line revealed that DIS3 is essential for cell survival, indicating that its function cannot be replaced by other exosome-associated nucleases: hDIS3L and hRRP6. Moreover, HEK293-derived cells, in which depletion of endogenous wild-type hDIS3 was complemented with exogenously expressed MM hDIS3 mutants, proliferate at a slower rate and exhibit aberrant RNA metabolism. Importantly, MM mutations are synthetically lethal with the hDIS3 PIN domain catalytic mutation both in yeast and human cells. Since mutations in PIN domain alone have little effect on cell physiology, our results predict the hDIS3 PIN domain as a potential drug target for MM patients with hDIS3 mutations. It is an interesting example of intramolecular synthetic lethality with putative therapeutic potential in humans.

Micro-organisms react to a rapid temperature downshift by triggering a physiological response to ensure survival in unfavourable conditions. Adaptation includes changes in membrane composition and in the translation and transcription machineries. The cold shock response leads to a growth block and overall repression of translation; however, there is the induction of a set of specific proteins that help to tune cell metabolism and readjust it to the new conditions. For a mesophile like E. coli, the adaptation process takes about 4 h. Although the bacterial cold shock response was discovered over two decades ago we are still far from understanding this process. In this review, we aim to describe current knowledge, focusing on the functions of RNA-interacting proteins and RNases involved in cold shock adaptation.

Objective: Mutations in the α-synuclein-encoding gene SNCA are considered as a rare cause of Parkinson's disease (PD). Our objective was to examine the frequency of the SNCA point mutations among PD patients of Polish origin., Methods: Detection of the known SNCA point mutations A30P (c.88G>C), E46K (c.136G>A) and A53T (c.157A>T) was performed either using the Sequenom MassArray iPLEX platform or by direct sequencing of the SNCA exons 2 and 3. As the two novel substitutions A18T (c.52G>A) and A29S (c.85G>T) were identified, their frequency in a control population of Polish origin was assessed and in silico analysis performed to investigate the potential impact on protein structure and function., Results: We did not observe the previously reported point mutations in the SNCA gene in our 629 PD patients; however, two novel potentially pathogenic substitutions A18T and A29S were identified. Each variant was observed in a single patient presenting with a typical late-onset sporadic PD phenotype. Although neither variant was observed in control subjects and in silico protein analysis predicts a damaging effect for A18T and pA29S substitutions, the lack of family history brings into question the true pathogenicity of these rare variants., Conclusions: Larger population based studies are needed to determine the pathogenicity of the A18T and A29S substitutions. Our findings highlight the possible role of rare variants contributing to disease risk and may support further screening of the SNCA gene in sporadic PD patients from different populations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

SGT1 (Suppressor of G2 allele of SKP1) is required to maintain plant disease Resistance (R) proteins with Nucleotide-Binding (NB) and Leucine-Rich Repeat (LRR) domains in an inactive but signaling-competent state. SGT1 is an integral component of a multi-protein network that includes RACK1, Rac1, RAR1, Rboh, HSP90 and HSP70, and in rice the Mitogen-Activated Protein Kinase (MAPK), OsMAPK6. Tobacco (Nicotiana tabacum) N protein, which belongs to the Toll-Interleukin Receptor (TIR)-NB-LRR class of R proteins, confers resistance to Tobacco Mosaic Virus (TMV). Following transient expression in planta, we analyzed the functional relationship between SGT1, SIPK - a tobacco MAPK6 ortholog - and N, using mass spectrometry, confocal microscopy and pathogen assays. Here, we show that tobacco SGT1 undergoes specific phosphorylation in a canonical MAPK target-motif by SIPK. Mutation of this motif to mimic SIPK phosphorylation leads to an increased proportion of cells displaying SGT1 nuclear accumulation and impairs N-mediated resistance to TMV, as does phospho-null substitution at the same residue. Forced nuclear localization of SGT1 causes N to be confined to nuclei. Our data suggest that one mode of regulating nucleocytoplasmic partitioning of R proteins is by maintaining appropriate levels of SGT1 phosphorylation catalyzed by plant MAPK., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Differential regulation of transcript stability is an effective means by which an organism can modulate gene expression. A well-characterized example is glutamine signalled degradation of specific transcripts in Aspergillus nidulans. In the case of areA, which encodes a wide-domain transcription factor mediating nitrogen metabolite repression, the signal is mediated through a highly conserved region of the 3' UTR. Utilizing this RNA sequence we isolated RrmA, an RNA recognition motif protein. Disruption of the respective gene led to loss of both glutamine signalled transcript degradation as well as nitrate signalled stabilization of niaD mRNA. However, nitrogen starvation was shown to act independently of RrmA in stabilizing certain transcripts. RrmA was also implicated in the regulation of arginine catabolism gene expression and the oxidative stress responses at the level of mRNA stability. ΔrrmA mutants are hypersensitive to oxidative stress. This phenotype correlates with destabilization of eifE and dhsA mRNA. eifE encodes eIF5A, a translation factor within which a conserved lysine is post-translationally modified to hypusine, a process requiring DhsA. Intriguingly, for specific transcripts RrmA mediates both stabilization and destabilization and the specificity of the signals transduced is transcript dependent, suggesting it acts in consort with other factors which differ between transcripts., (© 2013 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry-based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination-based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Sm-like (Lsm) proteins have been identified in all organisms and are related to RNA metabolism. Here, we report that Arabidopsis nuclear AtLSM8 protein, as well as AtLSM5, which localizes to both the cytoplasm and nucleus, function in pre-mRNA splicing, while AtLSM5 and the exclusively cytoplasmic AtLSM1 contribute to 5'-3' mRNA decay. In lsm8 and sad1/lsm5 mutants, U6 small nuclear RNA (snRNA) was reduced and unspliced mRNA precursors accumulated, whereas mRNA stability was mainly affected in plants lacking AtLSM1 and AtLSM5. Some of the mRNAs affected in lsm1a lsm1b and sad1/lsm5 plants were also substrates of the cytoplasmic 5'-3' exonuclease AtXRN4 and of the decapping enzyme AtDCP2. Surprisingly, a subset of substrates was also stabilized in the mutant lacking AtLSM8, which supports the notion that plant mRNAs are actively degraded in the nucleus. Localization of LSM components, purification of LSM-interacting proteins as well as functional analyses strongly suggest that at least two LSM complexes with conserved activities in RNA metabolism, AtLSM1-7 and AtLSM2-8, exist also in plants.

The multisubunit RNA exosome complex is a major ribonuclease of eukaryotic cells that participates in the processing, quality control and degradation of virtually all classes of RNA in Eukaryota. All this is achieved by about a dozen proteins with only three ribonuclease activities between them. At first glance, the versatility of the pathways involving the exosome and the sheer multitude of its substrates are astounding. However, after fifteen years of research we have some understanding of how exosome activity is controlled and applied inside the cell. The catalytic properties of the eukaryotic exosome are fairly well described and attention is now drawn to how the interplay between these activities impacts cell physiology. Also, it has become evident that exosome function relies on many auxiliary factors, which are intensely studied themselves. In this way, the focus of exosome research is slowly leaving the test tube and moving back into the cell. The exosome also has an interesting evolutionary history, which is evident within the eukaryotic lineage but only fully appreciated when considering similar protein complexes found in Bacteria and Archaea. Thus, while we keep this review focused on the most comprehensively described yeast and human exosomes, we shall point out similarities or dissimilarities to prokaryotic complexes and proteins where appropriate. The article is divided into three parts. In Part One we describe how the exosome is built and how it manifests in cells of different organisms. In Part Two we detail the enzymatic properties of the exosome, especially recent data obtained for holocomplexes. Finally, Part Three presents an overview of the RNA metabolism pathways that involve the exosome. This article is part of a Special Issue entitled: RNA Decay mechanisms., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Although the human mitochondrial genome has been investigated for several decades, the proteins responsible for its replication and expression, especially nucleolytic enzymes, are poorly described. Here, we characterized a novel putative PD-(D/E)XK nuclease encoded by the human C20orf72 gene named Ddk1 for its predicted catalytic residues. We show that Ddk1 is a mitochondrially localized metal-dependent DNase lacking detectable ribonuclease activity. Ddk1 degrades DNA mainly in a 3'-5' direction with a strong preference for single-stranded DNA. Interestingly, Ddk1 requires free ends for its activity and does not degrade circular substrates. In addition, when a chimeric RNA-DNA substrate is provided, Ddk1 can slide over the RNA fragment and digest DNA endonucleolytically. Although the levels of the mitochondrial DNA are unchanged on RNAi-mediated depletion of Ddk1, the mitochondrial single-stranded DNA molecule (7S DNA) accumulates. On the other hand, overexperssion of Ddk1 decreases the levels of 7S DNA, suggesting an important role of the protein in 7S DNA regulation. We propose a structural model of Ddk1 and discuss its similarity to other PD-(D/E)XK superfamily members.