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.