Non-microcystin extracellular metabolites of Microcystis aeruginosa impair viability and reproductive gene expression in rainbow trout cell lines.

Microcystis aeruginosa is a ubiquitous freshwater cyanobacterium best known for producing hepatotoxic microcystins; however, this common bloom-forming species also produces myriad biologically active and potentially deleterious other metabolites. Our understanding of the effects of these non-microcystin metabolites on fish is limited. In this study, we evaluated cytotoxicity of extracellular metabolites harvested from both microcystin-producing (MC+) and non-producing (MC-) strains of M. aeruginosa on rainbow trout (Oncorhynchus mykiss) cell lines derived from tissues of the brain, pituitary, heart, gonads, gills, skin, liver, and milt. We also examined the influence of M. aeruginosa exudates (MaE) on the expression of critical reproduction-related genes using the same cell lines. We found that exudates of the MC- M. aeruginosa strain significantly reduced viability in RTBrain, RTgill-W1, and RT-milt5 cell lines and induced significant cellular stress and/or injury in six of the eight cell lines-highlighting potential target tissues of cyanobacterial cytotoxic effects. Observed sublethal consequences of Microcystis bloom exposure occurred with both MC+ and MC- strains' exudates and significantly altered expression of developmental and sex steroidogenic genes. Collectively, our results emphasize the contributions of non-MC metabolites to toxicity of Microcystis-dominated algal blooms and the need to integrate the full diversity of M. aeruginosa compounds-beyond microcystins-into ecotoxicological risk assessments.

Characterization of natural antisense transcripts arisen from the locus encoding Toxoplasma gondii ubiquitin-like protease.

Natural antisense transcripts (NATs) are non-protein coding RNAs that could play an important role in regulating the expression of their counterpart protein encoding sense transcript. Although NATs are widespread in most eukaryotic genomes, very little is known about their functions. This study focuses on gaining a better understanding of the function of NATs in Toxoplasma gondii, a pathogenic unicellular eukaryote. Previously, we characterized the gene encoding the first committed enzyme in sumoylation, named ubiquitin-like protease 1 (TgUlp1), and showed that the expression of TgUlp1 is vital to the life cycle of T. gondii. Interestingly, the locus of TgUlp1 also transcribes a NAT species. Using a dual luciferase assay, we identified the promoter of TgUlp1 NAT to be located within the 3'-region of its counterpart coding sequence. While TgUlp1 mRNA level was detected at a lower level throughout the life cycle of T. gondii, its NAT level was upregulated when the parasite converts from actively replicating tachyzoite form to slowly growing bradyzoite form. To investigate the effect of TgUlp1 NAT on the expression of its counterpart mRNA, we used a reporter system bearing TgUlp1 mRNA sequences and showed that the single-stranded TgUlp1 NAT and its in vitro RNase III processed products have the ability to lower the expression of the reporter system. Using a transgenic Dicer-knockout (TgDicer-KO) strain, we showed that TgDicer is required for the function of TgUlp1 NAT in vivo. The findings strongly suggest that the RNA interference pathway is necessary for the function of TgUlp1 NAT.

Toxoplasma ubiquitin-like protease 1, a key enzyme in sumoylation and desumoylation pathways, is under the control of non-coding RNAs.

Sumoylation and desumoylation are reversible pathways responsible for modification of protein structures and functions by the reversible covalent attachment of a small ubiquitin-like modifier (SUMO) peptide. These pathways are important for a wide range of cellular processes and require a steady supply of SUMO, which is generated by an enzymatic reaction catalysed by the ubiquitin-like protease (Ulp) family. Here we show by functional complementation analysis that the Ulp1 of Toxoplasma gondii (TgUlp1) can rescue a growth-deficient phenotype of a yeast-Ulp1 knockout. Recombinant TgUlp1 is an active enzyme capable of removing SUMO from a sumoylated substrate. Using a clonal transgenic strain of T. gondii expressing an epitope-tagged version of TgUlp1, we detected that the expression of TgUlp1 is modulated by Tg-miR-60, the most abundant species of micro RNA found in the T. gondii type 1 strain. The introduction of Tg-miR-60 inhibitor caused an increase in TgUlp1 expression and its enzymatic activity, as well as affecting the parasite's growth fitness. Moreover, we discovered a polyadenylated antisense RNA transcribed from the TgUlp1 locus, referred to as TgUlp1-NAT1 (TgUlp1-natural antisense transcript 1). Both Tg-miR-60 and TgUlp1-NAT1 confer a regulatory function by down-regulating the expression of TgUlp1 and affecting the sumoylation and desumoylation pathways in T. gondii.