Trypanosomes are parasitic protozoans that include several medically and a variety of economically important parasites, such as Trypanosoma brucei, the causative agent of sleeping sickness. This parasite cycles between the insect host (procyclic form) and mammalian host (bloodstream form). These parasites lack transcription regulation, including factors that govern the unfolded protein response (UPR) in other eukaryotes. Gene expression is controlled posttranscriptionally by unique mechanisms such as trans-splicing and RNA editing and by mRNA stability. In trans-splicing, a common exon, the spliced leader (SL) is donated to all mRNAs from a small RNA, the SL RNA. The SL RNA is transcribed from a defined promoter assisted by the tSNAP complex. Despite the lack of transcriptional regulation, induction of ER stress elicits changes in the transcriptome similar to those induced by conventional UPR found in other eukaryotes. The mechanism of upregulation under UPR is dependent on differential stabilization of mRNAs. The transcriptome changes result in ER expansion and elevation in the ER chaperone, BiP. Prolonged ER stress induces the spliced leader RNA silencing (SLS) pathway. SLS is the trypanosome-specific stress response mechanism that elicits the shut-off of SL RNA transcription by perturbing the binding of the transcription factor tSNAP42 to its cognate promoter, eliminating trans-splicing of all mRNAs. SLS was discovered in the RNAi silenced cells depleted for functions that mediate translocation of proteins to the ER such as the signal recognition particle receptor SRα, SEC63 - a factor that participates in protein translocation across the ER membrane, or SEC61 - the translocation channel. Induction of SLS, either by prolonged ER stress or silencing of the genes associated with the ER membrane that function in ER protein translocation led to programmed cell death (PCD), evident by the exposure of phosphatidyl serine, DNA laddering, increase in ROS production, increase in cytoplasmic Ca 2+ , and decrease in mitochondrial membrane potential. Here, we describe the protocols to induce ER stress and to observe the resulting morphological changes by transmission electron microscopy (TEM), changes in cytoplasmic Ca 2+ , and DNA fragmentation which are the hallmarks of programmed cell death.
|Title of host publication||Methods in Enzymology|
|Publisher||Academic Press Inc.|
|Number of pages||17|
|State||Published - 2011|
|Name||Methods in Enzymology|
Bibliographical noteFunding Information:
The research relevant to this work was supported by a grant from the Israel Science Foundation (ISF) and by an International Research Scholar's Grant from the Howard Hughes Foundation to S.M. S.M. holds the David and Inez Myers Chair in RNA silencing of diseases.