Gene organization and sequence analyses of transfer RNA genes in Trypanosomatid parasites

<p>Abstract</p> <p>Background</p> <p>The protozoan pathogens <it>Leishmania major</it>, <it>Trypanosoma brucei </it>and <it>Trypanosoma cruzi </it>(the Tritryps) are parasites that produce devastating human diseases. These organisms show very unusual mechanisms of gene expression, such as polycistronic transcription. We are interested in the study of tRNA genes, which are transcribed by RNA polymerase III (Pol III). To analyze the sequences and genomic organization of tRNA genes and other Pol III-transcribed genes, we have performed an <it>in silico </it>analysis of the Tritryps genome sequences.</p> <p>Results</p> <p>Our analysis indicated the presence of 83, 66 and 120 genes in <it>L. major, T. brucei </it>and <it>T. cruzi</it>, respectively. These numbers include several previously unannotated selenocysteine (Sec) tRNA genes. Most tRNA genes are organized into clusters of 2 to 10 genes that may contain other Pol III-transcribed genes. The distribution of genes in the <it>L. major </it>genome does not seem to be totally random, like in most organisms. While the majority of the tRNA clusters do not show synteny (conservation of gene order) between the Tritryps, a cluster of 13 Pol III genes that is highly syntenic was identified. We have determined consensus sequences for the putative promoter regions (Boxes A and B) of the Tritryps tRNA genes, and specific changes were found in tRNA-Sec genes. Analysis of transcription termination signals of the tRNAs (clusters of Ts) showed differences between <it>T. cruzi </it>and the other two species. We have also identified several tRNA isodecoder genes (having the same anticodon, but different sequences elsewhere in the tRNA body) in the Tritryps.</p> <p>Conclusion</p> <p>A low number of tRNA genes is present in Tritryps. The overall weak synteny that they show indicates a reduced importance of genome location of Pol III genes compared to protein-coding genes. The fact that some of the differences between isodecoder genes occur in the internal promoter elements suggests that differential control of the expression of some isoacceptor tRNA genes in Tritryps is possible. The special characteristics found in Boxes A and B from tRNA-Sec genes from Tritryps indicate that the mechanisms that regulate their transcription might be different from those of other tRNA genes.</p

Associations between whole peripheral blood fatty acids and DNA methylation in humans

Fatty acids (FA) modify DNA methylation in vitro, but limited information is available on whether corresponding associations exist in vivo and reflect any short-term effect of the diet. Associations between global DNA methylation and FAs were sought in blood from lactating infants (LI; n = 49) and adult males (AMM; n = 12) equally distributed across the three conventional BMI classes. AMM provided multiple samples at 2-hour intervals during 8 hours after either a single Western diet-representative meal (post-prandial samples) or no meal (fasting samples). Lipid/glucose profile, HDAC4 promoter and PDK4 5'UTR methylation were determined in AMM. Multiple regression analysis revealed that global (in LI) and both global and PDK4-specific DNA methylation (in AMM) were positively associated with eicosapentaenoic and arachidonic acid. HDAC4 methylation was inversely associated with arachidonic acid post-prandially in AMM. Global DNA methylation did not show any defined within-day pattern that would suggest a short-term response to the diet. Nonetheless, global DNA methylation was higher in normal weight subjects both post-prandially and in fasting and coincided with higher polyunsaturated relative to monounsaturated and saturated FAs. We show for the first time strong associations of DNA methylation with specific FAs in two human cohorts of distinct age, diet and postnatal development stage

Effect of recombinant TCTP on B cell proliferation.

<p>Splenic B cells (2×10⁶cells/well) were plated with RPMI 1640 supplemented with 10% FBS and incubated with 10 µg/mL of recombinant HsTCTP or PfTCTP labeled with Oregon Green 488 (green) fluorochrome for 24–120 h at 37°C and 5% CO₂. LPS (1 µg/mL) was used as positive control. Untreated and 20 mM phosphate buffer-treated B cells were used as negative controls. Viable B cell density was measured with a Countess Automated Cell Counter. Results are representative of two independent experiments and are expressed as the mean ±SD of duplicate cultures. *, <i>p</i><0.05 vs 10 µg/mL labeled PfTCTP treated B cells.</p

Effect of recombinant TCTP in the progression of cell cycle from B cells.

<p>Splenic B cells (2×10⁶cells/well) were plated with RPMI 1640 supplemented with 10% FBS and incubated with 10 µg/mL of recombinant HsTCTP or PfTCTP labeled with Oregon Green 488 (green) fluorochrome for 24–120 h at 37°C and 5% CO₂. The percentage of cell cycle distribution and incorporation of fluorescently-labeled of PfTCTP and HsTCTP into splenic B cells was analyzed by flow cytometry (FACS). Results (%) are expressed as mean ± SD of duplicate determinations. One of two similar experiments is shown. Incorporation, percentage of incorporation of fluorescently-labeled protein into B cells.</p><p>*, <i>p</i><0.05 vs 10 µg/mL labeled HsTCTP treated B cells</p><p>**, <i>p</i><0.01 vs 10 µg/mL labeled HsTCTP treated B cells</p

Localization of incorporated TCTP in B cells.

<p>Confocal microscopy analysis of negatively isolated B cells (2×10⁵cells/well) incubated 30 min with 50 ng/mL of recombinant HsTCTP labeled with Oregon Green 488 dye (green) and 50 ng/mL of recombinant PfTCTP labeled with Alexa Fluor 594 dye (red). Representative images are shown. A. Bright field. B. DAPI staining C. Green fluorescence. D. Red fluorescence. E. Merge.</p

In Silico Identification and Characterization of circRNAs as Potential Virulence-Related miRNA/siRNA Sponges from Entamoeba histolytica and Encystment-Related circRNAs from Entamoeba invadens

Ubiquitous eukaryotic non-coding circular RNAs regulate transcription and translation. We have reported full-length intronic circular RNAs (flicRNAs) in Entamoeba histolytica with esterified 3&prime;ss and 5&prime;ss. Their 5&prime;ss GU-rich elements are essential for their biogenesis and their suggested role in transcription regulation. Here, we explored whether exonic, exonic-intronic, and intergenic circular RNAs are also part of the E. histolytica and E. invadens ncRNA RNAome and investigated their possible functions. Available RNA-Seq libraries were analyzed with the CIRI-full software in search of circular exonic RNAs (circRNAs). The robustness of the analyses was validated using synthetic decoy sequences with bona fide back splice junctions. Differentially expressed (DE) circRNAs, between the virulent HM1:IMSS and the nonvirulent Rahman E. histolytica strains, were identified, and their miRNA sponging potential was analyzed using the intaRNA software. Respectively, 188 and 605 reverse overlapped circRNAs from E. invadens and E. histolytica were identified. The sequence composition of the circRNAs was mostly exonic although different to human circRNAs in other attributes. 416 circRNAs from E. histolytica were virulent-specific and 267 were nonvirulent-specific. Out of the common circRNAs, 32 were DE between strains. Finally, we predicted that 8 of the DE circRNAs could function as sponges of the bioinformatically reported miRNAs in E. histolytica, whose functions are still unknown. Our results extend the E. histolytica RNAome and allow us to devise a hypothesis to test circRNAs/miRNAs/siRNAs interactions in determining the virulent/nonvirulent phenotypes and to explore other regulatory mechanisms during amoebic encystment

Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

The fastq files supporting the results of this article are available in the EMBL‐EBI European Nucleotide Archive (ENA: PRJEB18114; Sample group: ERG011046): http://www.ebi.ac.uk/ena/data/view/PRJEB18114.International audiencePost‐translational modifications of histone H3 N‐terminal tails are key epigenetic regulators of virulence gene expression and sexual commitment in the human malaria parasite Plasmodium falciparum. Here, we identify proteolytic clipping of the N‐terminal tail of nucleosome‐associated histone H3 at amino acid position 21 as a new chromatin modification. A cathepsin C‐like proteolytic clipping activity is observed in nuclear parasite extracts. Notably, an ectopically expressed version of clipped histone H3, PfH3p‐HA, is targeted to the nucleus and integrates into mononucleosomes. Furthermore, chromatin immunoprecipitation and next‐generation sequencing analysis identified PfH3p‐HA as being highly enriched in the upstream region of six genes that play a key role in DNA replication and repair: In these genes, PfH3p‐HA demarcates a specific 1.5 kb chromatin island adjacent to the open reading frame. Our results indicate that, in P. falciparum, the process of histone clipping may precede chromatin integration hinting at preferential targeting of pre‐assembled PfH3p‐containing nucleosomes to specific genomic regions. The discovery of a protease‐directed mode of chromatin organization in P. falciparum opens up new avenues to develop new anti‐malarials

Additional file 1: Figure S1. of Identification of repressive and active epigenetic marks and nuclear bodies in Entamoeba histolytica

The N-terminal region of histone H4 of E. histolytica is conserved among different eukaryotic cells. A) Alignment of the N-terminal region of histone H4 from E. histolytica (Eh), P. falciparum (Pf), Saccharomyces cerevisiae (Sc), Drosophila melanogaster (Dm), and Homo sapiens (Hs). (*). Identical residues; (:) compensatory changes. Three insertions present in the N-terminal region of histone H4 of E. histolytica are indicated in red letters. B) Elimination of the three insertions present in the N-terminal region of histone H4 of Eh (indicated by lines) generates a highly conserved N-terminal region among Eh, Pf, Sc, Dm and Hs. Alignment of the H4 N-terminal region of Eh, Pf, Sc, Dm and Hs with H4 pan-acetylated peptide from Tetrahymena thermophiles and a H4 arginine 3 mono-methylated peptide are shown. Rectangle indicated the most conserved residues identified for both peptides. Lysines and arginine amino acids susceptible to be acetylated are indicated in bold. (DOCX 27 kb