MicroRNAs (miRNAs) are small noncoding RNA molecules that negatively control expression of target genes in animals and plants. The microRNA-21 gene (mir-21) has been identified as the only miRNA commonly overexpressed in solid tumors of the lung, breast, stomach, prostate, colon, brain, head and neck, esophagus and pancreas. We initiated a screen to identify miR-21 target genes using a reporter assay and identified a potential miR-21 target in the 3 0 -UTR of the programmed cell death 4 (PDCD4) gene. We cloned the full-length 3 0 -UTR of human PDCD4 downstream of a reporter and found that mir-21 downregulated, whereas a modified antisense RNA to miR-21 upregulated reporter activity. Moreover, deletion of the putative miR-21-binding site (miRNA regulatory element, MRE) from the 3 0 -UTR of PDCD4, or mutations in the MRE abolished the ability of miR-21 to inhibit reporter activity, indicating that this MRE is a critical regulatory region. Western blotting showed that Pdcd4 protein levels were reduced by miR-21 in human and mouse cells, whereas quantitative real-time PCR revealed little difference at the mRNA level, suggesting translational regulation. Finally, overexpression of mir-21 in MCF-7 human breast cancer cells and mouse epidermal JB6 cells promoted soft agar colony formation by downregulating Pdcd4 protein levels. The demonstration that miR-21 promotes cell transformation supports the concept that mir-21 functions as an oncogene by a mechanism that involves translational repression of the tumor suppressor Pdcd4.

Misfolded alpha-synuclein (AS) and other neurodegenerative disorder proteins display prion-like transmission of protein aggregation. Factors responsible for the initiation of AS aggregation are unknown. To evaluate the role of amyloid proteins made by the microbiota we exposed aged rats and transgenic C. elegans to E. coli producing the extracellular bacterial amyloid protein curli. Rats exposed to curli-producing bacteria displayed increased neuronal AS deposition in both gut and brain and enhanced microgliosis and astrogliosis compared to rats exposed to either mutant bacteria unable to synthesize curli, or to vehicle alone. Animals exposed to curli producing bacteria also had more expression of TLR2, IL-6 and TNF in the brain than the other two groups. There were no differences among the rat groups in survival, body weight, inflammation in the mouth, retina, kidneys or gut epithelia, and circulating cytokine levels. AS-expressing C. elegans fed on curli-producing bacteria also had enhanced AS aggregation. These results suggest that bacterial amyloid functions as a trigger to initiate AS aggregation through cross-seeding and also primes responses of the innate immune system.

Long interspersed nuclear elements [LINE-1 (L1)] are abundant retrotransposons in mammalian genomes that remain silent under most conditions. Cellular stress signals activate L1, but the molecular mechanisms controlling L1 activation remain unclear. Evidence is presented here that benzo(a) pyrene (BaP), an environmental hydrocarbon metabolized by mammalian cytochrome P450s to reactive carcinogenic intermediates, increases L1 retrotransposition in HeLa cells. Increased retrotransposition is mediated by up-regulation of L1 RNA levels, increased L1 cDNA synthesis, and stable genomic integration. Activation of L1 is dependent on the ability of BaP to cause DNA damage because it is absent in HeLa cells challenged with nongenotoxic hydrocarbon carcinogens. Thus, the mutations and genomic instability observed in human populations exposed to genotoxic environmental hydrocarbons may involve epigenetic activation of mobile elements dispersed throughout the human genome.

The present study was conducted to evaluate the contextual specificity of long interspersed nuclear element-1 ( LINE-1 or L1 ) activation by cellular stress and the role of the aryl hydrocarbon receptor (AHR) transcription factor and oxidative stress in the gene activation response.

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (−10.1 ± 0.3 kcal mol−1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

Factor XIII A (FXIIIA) is a member of the transglutaminase enzyme family that cross-links both intra- and extracellular protein substrates. To prevent undesired cross-linking, FXIIIA is expressed as an inactive zymogen and exists intracellularly as an A2 homodimer. In plasma, FXIII A2 is complexed with two protective factor XIII B subunits (A2B2) that dissociate upon activation of the zymogen. Based on limited experimental data, activated FXIII was considered a dimer of two catalytically active A subunits. However, accumulating but indirect evidence has suggested activation may lead to a monomeric state instead. In the present study, we employed analytical ultracentrifugation (AUC) to directly explore the oligomerization state of zymogen as well as active FXIIIA in solution. We first confirmed that the zymogen was a FXIIIA2 dimer. When we activated FXIIIA non-proteolytically (by high mM Ca2+), the protein dissociated to monomers. More importantly, FXIIIA incubation with its physiological partner, the protease thrombin, led to a monomeric state as well. AUC studies of partially cleaved FXIIIA further suggested that thrombin cleavage of a single activation peptide in a zymogen dimer is sufficient to weaken inter-subunit interactions, initiating the transition to monomer. The enzymatic activity of the thrombin-cleaved species was higher than non-proteolytically activated enzyme, suggesting that displacement of the activation peptide renders the FXIIIA more accessible to substrates. Thus, results provide evidence that FXIII undergoes a change in oligomerization state as part of the activation process, and emphasize the role of the activation peptide in preventing FXIIIA catalytic activity.

We have found an extremely large ribonuclease P (RNase P) RNA (RPR1) in the human pathogen Candida glabrata and verified that this molecule is expressed and present in the active enzyme complex of this hemiascomycete yeast. A structural alignment of the C. glabrata sequence with 36 other hemiascomycete RNase P RNAs (abbreviated as P RNAs) allows us to characterize the types of insertions. In addition, 15 P RNA sequences were newly characterized by searching in the recently sequenced genomes Candida albicans, C. glabrata, Debaryomyces hansenii, Eremothecium gossypii, Kluyveromyces lactis, Kluyveromyces waltii, Naumovia castellii, Saccharomyces kudriavzevii, Saccharomyces mikatae, and Yarrowia lipolytica; and by PCR amplification for other Candida species (Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida stellatoidea, and Candida tropicalis). The phylogenetic comparative analysis identifies a hemiascomycete secondary structure consensus that presents a conserved core in all species with variable insertions or deletions. The most significant variability is found in C. glabrata P RNA in which three insertions exceeding in total 700 nt are present in the Specificity domain. This P RNA is more than twice the length of any other homologous P RNAs known in the three domains of life and is eight times the size of the smallest. RNase P RNA, therefore, represents one of the most diversified noncoding RNAs in terms of size variation and structural diversity.

RNase MRP is ae ukaryote-specific endoribonuclease that generates RNA primers for mitochondrial DNA replication and processes precursor rRNA. RNase Pi saubiquitous endoribonuclease that cleaves precursor tRNA transcripts to produce their mature 5 9 termini. We found extensive sequence homology of catalytic domains and specificity domains between their RNA subunits in many organisms. In Candida glabrata,t he internal loop of helix P3 is 100% conserved between MRP and PR NAs. The helix P8 of MRP RNA from microsporidia Encephalitozoon cuniculi is identical to that of PRNA. Sequence homology can be widely spread over the whole molecule of MRP RNA and PRNA, such as those from Dictyostelium discoideum.These conserved nucleotides between the MRP and PR NAs strongly support the hypothesis that the MRP RNA is derived from the PR NA molecule in early eukaryote evolution.Keywords: specificity domain; catalytic domain; RNase MRP RNA RNasePandR Nase MRPa re ribonucleoprotein (RNP) complexes participating in cellularR NA processing. RNase Pi saubiquitouse ndoribonucleaset hatc leavesp recursor tRNA (pre-tRNA) transcripts to produce their mature 5 9 termini (Altman1 990; Altmana nd Kirsebom 1999).B acterialR Nase Pc onsistso facatalytic RNA subunit and a protein cofactor,w hilea rchaeal and eukaryotic enzymes haveasingle RNA subunit andm oret hanf ourp roteins (Hartmann and Hartmann2003). RNase MRPisaeukaryotespecific endoribonuclease that has at least two roles: one in themitochondrial compartmentwhere it generates RNA primersfor theinitiationofmitochondrial DNAreplication (Chang and Clayton 1989), and as econd in the nucleolus whereitparticipates in precursorrRNAprocessing (Lygerou et al. 1996). Hemiascomycetes, such as the budding yeast Saccharomyces cerevisiae,havebeenextensively used to study the structureand functionofeukaryotic RNase Pand MRP. The S. cerevisiae RNase Penzyme possessesone RNA (P RNA) and nine integral protein subunits, while RNase MRP has one RNA (MRP RNA) and 10 proteins (Chamberlain et al. 1998;Salinas et al. 2005). Previously, the catalytic domains of these two RNAs havebeen demonstrated to havesimilar secondary structures (Li et al. 2002). Moreover, eight of the associated protein components are identical ( Chamberlain et al. 1998), indicating that these two enzymecomplexes are structurallya nd evolutionally related.Despite structural similarity in the catalytic domains, little sequence homology beyond the P3 and P4 helices of Pa nd MRP RNA has been demonstrated, partially due to limited availability of sequences, especially the MRP RNA sequences. Ar ecent report by Samuelsson and colleagues identified more than 100 new MRP and PR NA sequences frome ukaryotes with completeg enomes equences or whole-genome-shotguns equences (Piccinelli et al. 2005). Some of thesep redictedM RP and PR NA genes from Candida speciesare demonstrated by biochemical methods in our laboratory to be expressed (data not shown). These sequences allowed us to perform ac omprehensive analysis of MRP RNAs and their homolog...