The Trypanosoma brucei RNA-Binding Protein TbRRM1 is Involved in the Transcription of a Subset of RNA Pol II-Dependent Genes.

It has been long thought that RNA Polymerase (Pol) II transcriptional regulation does not operate in trypanosomes. However, recent reports have suggested that these organisms could regulate RNA Pol II transcription by epigenetic mechanisms. In this paper, we investigated the role of TbRRM1 in transcriptional regulation of RNA Pol II-dependent genes by focusing both in genes located in a particular polycistronic transcription unit (PTU) and in the monocistronic units of the SL-RNA genes. We showed that TbRRM1 is recruited throughout the PTU, with a higher presence on genes than intergenic regions. However, its depletion leads both to the decrease of nascent RNA and to chromatin compaction only of regions located distal to the main transcription start site. These findings suggest that TbRRM1 facilitates the RNA Pol II transcriptional elongation step by collaborating to maintain an open chromatin state in particular regions of the genome. Interestingly, the SL-RNA genes do not recruit TbRRM1 and, after TbRRM1 knockdown, nascent SL-RNAs accumulate while the chromatin state of these regions remains unchanged. Although it was previously suggested that TbRRM1 could regulate RNA Pol II-driven genes, we provide here the first experimental evidence which involves TbRRM1 to transcriptional regulation.

TbRRM1 knockdown produces abnormal cell morphology and apoptotic-like death in the bloodstream form of T. brucei.

TbRRM1, an SR-related protein, is involved in transcriptional and post-transcriptional gene expression regulation in procyclic T. brucei. In previous work, we found that TbRRM1 is essential and its depletion leads to cell cycle impairment, aberrant phenotypes and cell loss by apoptotic-like death. Here, we report the findings obtained after TbRRM1 knockdown in bloodstream parasites. Depletion of TbRRM1 in this cell stage led also to growth arrest and cell loss by apoptosis-like death. However, microscopic analysis showed aberrant cell morphology with parasites displaying flagellum detachment and cytokinesis impairment after RNAi induction, suggesting that TbRRM1 could play different roles depending on parasite stage.

Depletion of the SR-Related Protein TbRRM1 Leads to Cell Cycle Arrest and Apoptosis-Like Death in Trypanosoma brucei.

Arginine-Serine (RS) domain-containing proteins are RNA binding proteins with multiple functions in RNA metabolism. In mammalian cells this group of proteins is also implicated in regulation and coordination of cell cycle and apoptosis. In trypanosomes, an early branching group within the eukaryotic lineage, this group of proteins is represented by 3 members, two of them are SR proteins and have been recently shown to be involved in rRNA processing as well as in pre-mRNA splicing and stability. Here we report our findings on the 3rd member, the SR-related protein TbRRM1. In the present study, we showed that TbRRM1 ablation by RNA-interference in T. brucei procyclic cells leads to cell-cycle block, abnormal cell elongation compatible with the nozzle phenotype and cell death by an apoptosis-like mechanism. Our results expand the role of the trypanosomal RS-domain containing proteins in key cellular processes such as cell cycle and apoptosis-like death, roles also carried out by the mammalian SR proteins, and thus suggesting a conserved function in this phylogenetically conserved protein family.

Antiviral action of synthetic stigmasterol derivatives on herpes simplex virus replication in nervous cells in vitro.

Polyfunctionalized stigmasterol derivatives, (22S,23S)-22,23-dihydroxystigmast-4-en-3-one (compound 1) and (22S,23S)-3ß-bromo-5α,22,23-trihydroxystigmastan-6-one (compound 2), inhibit herpes simplex virus type 1 (HSV-1) replication and spreading in human epithelial cells derived from ocular tissues. Both compounds reduce the incidence and severity of lesions in a murine model of herpetic stromal keratitis when administered in different treatment modalities. Since encephalitis caused by HSV-1 is another immunopathology of viral origin, we evaluate here the antiviral effect of both compounds on HSV-1 infected nervous cell lines as well as their anti-inflammatory action. We found that both stigmasterol derivatives presented low cytotoxicity in the three nervous cell lines assayed. Regarding the antiviral activity, in all cases both compounds prevented HSV-1 multiplication when added after infection, as well as virus propagation. Additionally, both compounds were able to hinder interleukin-6 and Interferon-gamma secretion induced by HSV-1 infection in Neuro-2a cells. We conclude that compounds 1 and 2 have exerted a dual antiviral and anti-inflammatory effect in HSV-1 infected nervous cell lines, which makes them interesting molecules to be further studied.