The mechanism and machinery of spliced leader RNA silencing (SLS), a stress induced mechanism leading to programmed cell death in Trypanosoma brucei

Abstract

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, trypanosomes possess a stress response mechanism, the spliced leader RNA (SL RNA) silencing (SLS) pathway. SLS is induced under prolonged ER stress or when translocation to the ER is perturbed upon RNAi silencing of factors involved in protein translocation across the ER membrane such as (SRP receptor, and the ER translocation factors SEC63 or SEC61). SLS leads to shutoff of SL RNA transcription by avoiding the binding of the SL RNA‐specific transcription factor tSNAP42 to its cognate promoter (Lustig al., EMBO Rep. 2007). Since SL RNA donates the SL exon to all mRNAs via trans‐splicing, SLS induction inhibits mRNA production. Induction of SLS leads to programmed cell death (PCD), evident by exposure of phosphatidyl serine and DNA laddering (Goldshmidt et al., PLoS Pathogens, 2010). 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. To investigate the mechanism of SL RNA transcription shut‐off during SLS, the SL RNA transcription complex was purified under SLS and mass‐spectrometry analysis indicated unique phosphorylation of the TATA‐binding protein TBP. A PERK‐like kinase was shown to be involved in SLS signaling, and its silencing abolished SLS induction. Using iTRAQ analysis we identified proteins that their level was elevated during SLS and focused on two proteases; rhomboid and calpain‐like proteases. Silencing of calpain‐like protease abolished SLS. I will discuss our approaches to explore the sequential events and the interplay between the different cascades leading to SLS and PCD.