Persistent ER Stress Induces the Spliced Leader RNA Silencing Pathway (SLS), Leading to Programmed Cell Death in Trypanosoma brucei

Trypanosomes are parasites that cycle between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack conventional transcription regulation, including factors that induce the unfolded protein response (UPR). However, they possess a stress response mechanism, the spliced leader RNA silencing (SLS) pathway. SLS elicits shut-off of spliced leader RNA (SL RNA) transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, thus eliminating trans-splicing of all mRNAs. Induction of endoplasmic reticulum (ER) stress in procyclic trypanosomes elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of up-regulation under ER stress is dependent on differential stabilization of mRNAs. The transcriptome changes are accompanied by ER dilation and elevation in the ER chaperone, BiP. Prolonged ER stress induces SLS pathway. RNAi silencing of SEC63, a factor that participates in protein translocation across the ER membrane, or SEC61, the translocation channel, also induces SLS. Silencing of these genes or prolonged ER stress led to programmed cell death (PCD), evident by exposure of phosphatidyl serine, DNA laddering, increase in reactive oxygen species (ROS) production, increase in cytoplasmic Ca2+, and decrease in mitochondrial membrane potential, as well as typical morphological changes observed by transmission electron microscopy (TEM). ER stress response is also induced in the bloodstream form and if the stress persists it leads to SLS. We propose that prolonged ER stress induces SLS, which serves as a unique death pathway, replacing the conventional caspase-mediated PCD observed in higher eukaryotes

The stage of soil development modulates rhizosphere effect along a High Arctic desert chronosequence

In mature soils, plant species and soil type determine the selection of root microbiota. Which of these two factors drives rhizosphere selection in barren substrates of developing desert soils has, however, not yet been established. Chronosequences of glacier forelands provide ideal natural environments to identify primary rhizosphere selection factors along the changing edaphic conditions of a developing soil. Here, we analyze changes in bacterial diversity in bulk soils and rhizospheres of a pioneer plant across a High Arctic glacier chronosequence. We show that the developmental stage of soil strongly modulates rhizosphere community assembly, even though plant-induced selection buffers the effect of changing edaphic factors. Bulk and rhizosphere soils host distinct bacterial communities that differentially vary along the chronosequence. Cation exchange capacity, exchangeable potassium, and metabolite concentration in the soil account for the rhizosphere bacterial diversity. Although the soil fraction (bulk soil and rhizosphere) explains up to 17.2% of the variation in bacterial microbiota, the soil developmental stage explains up to 47.7% of this variation. In addition, the operational taxonomic unit (OTU) co-occurrence network of the rhizosphere, whose complexity increases along the chronosequence, is loosely structured in barren compared with mature soils, corroborating our hypothesis that soil development tunes the rhizosphere effect

snRNA-specific role of SMN in trypanosome snRNP biogenesis in vivo

Pre-mRNA splicing in trypanosomes requires the SMN-mediated assembly of small nuclear ribonucleoproteins (snRNPs). In contrast to higher eukaryotes, the cellular localization of snRNP biogenesis and the involvement of nuclear-cytoplasmic trafficking in trypanosomes are controversial. By using RNAi knockdown of SMN in T. brucei to investigate its functional role in snRNP assembly, we found dramatic changes in the steady-state levels of snRNAs and snRNPs: The SL RNA accumulates, whereas U1, U4 and U5 snRNA levels decrease, and Sm core assembly in particular of the SL RNA is strongly reduced. In addition, SMN depletion blocks U4/U6 di-snRNP formation; the variant Sm core of the U2 snRNP, however, still forms efficiently after SMN knockdown. Concerning the longstanding question, whether nuclear-cytoplasmic trafficking is involved in trypanosomal snRNP biogenesis, fluorescence in situ hybridization (FISH) and immunofluorescence assays revealed that the SL RNA genes and transcripts colocalize with SMN. Remarkably, SMN silencing leads to a nucleoplasmic accumulation of both SL RNA and the Sm proteins. In sum, our data demonstrate an essential and snRNA-selective role of SMN in snRNP biogenesis in vivo and strongly argue for a nucleoplasmic Sm core assembly of the SL RNP

New insights into trypanosomatid U5 small nuclear ribonucleoproteins

Several protozoan parasites exist in the Trypanosomatidae family, including various agents of human diseases. Multiple lines of evidence suggest that important differences are present between the translational and mRNA processing (trans splicing) systems of trypanosomatids and other eukaryotes. In this context, certain small complexes of RNA and protein, which are named small nuclear ribonucleoproteins (U snRNPs), have an essential role in pre-mRNA processing, mainly during splicing. Even though they are well defined in mammals, snRNPs are still not well characterized in trypanosomatids. This study shows that a U5-15K protein is highly conserved among various trypanosomatid species. Tandem affinity pull-down assays revealed that this protein interacts with a novel U5-102K protein, which suggests the presence of a sub-complex that is potentially involved in the assembly of U4/U6-U5 tri-snRNPs. Functional analyses showed that U5-15K is essential for cell viability and is somehow involved with the trans and cis splicing machinery. Similar tandem affinity experiments with a trypanonosomatid U5-Cwc21 protein led to the purification of four U5 snRNP specific proteins and a Sm core, suggesting U5-Cwc-21 participation in the 35S U5 snRNP particle. Of these proteins, U5-200K was molecularly characterized. U5-200K has conserved domains, such as the DEAD/DEAH box helicase and Sec63 domains and displays a strong interaction with U5 snRNA