Spliced leader RNA silencing (SLS) - a programmed cell death pathway in Trypanosoma brucei that is induced upon ER stress

Abstract

Trypanosoma brucei is the causative agent of African sleeping sickness. The parasite cycles between its insect (procyclic form) and mammalian hosts (bloodstream form). Trypanosomes lack conventional transcription regulation, and their genes are transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. In trans-splicing, which is essential for processing of each mRNA, an exon, the spliced leader (SL) is added to all mRNAs from a small RNA, the SL RNA. Trypanosomes lack the machinery for the unfolded protein response (UPR), which in other eukaryotes is induced under endoplasmic reticulum (ER) stress. Trypanosomes respond to such stress by changing the stability of mRNAs, which are essential for coping with the stress. However, under severe ER stress that is induced by blocking translocation of proteins to the ER, treatment of cells with chemicals that induce misfolding in the ER, or extreme pH, trypanosomes elicit the spliced leader silencing (SLS) pathway. In SLS, the transcription of the SL RNA gene is extinguished, and tSNAP42, a specific SL RNA transcription factor, fails to bind to its cognate promoter. SLS leads to complete shut-off of trans-splicing. In this review, I discuss the UPR in mammals and compare it to the ER stress response in T. brucei leading to SLS. I summarize the evidence supporting the notion that SLS is a programmed cell death (PCD) pathway that is utilized by the parasites to substitute for the apoptosis observed in higher eukaryotes under prolonged ER stress. I present the hypothesis that SLS evolved to expedite the death process, and rapidly remove from the population unfit parasites that, by elimination via SLS, cause minimal damage to the parasite population.

Figure 1

The ER-quality control. Upon translocation to the ER the N-glycan is ligated to the nascent chain. Then two glucosidases I and II remove glucose group. The mono-glucosylated glycoprotein then interacts with calnexin/calreticulin. These chaperones recruit the oxireductase ERp57. Cleavage of the last glucose residue by glucosidase II leads to the release of chaperones. At this stage if the protein is properly folded it will exit the ER. The incorrectly folded protein is the substrate of UDP/glucose:glycoprotein glucosyltransferase, which puts glucose back to the misfolded protein. If the protein fails to fold properly even after several cycles, the manose residue is removed by the mannosidase I. This modified glycan is recognized by the (ER degradation enhancing mannosidase-like protein) (EDEM). This targets the misfolded protein for ER-associated degradation (ERAD). The factors missing in trypanosomes but exist in other eukaryotes are crossed.

Figure 2

The two branches of the unfolded protein response. As a result of accumulation of misfolded proteins in the ER, the unfolded protein response is initiated. Three signal transduction pathways coordinate the pathway and require the dissociation of the ER chaperone BiP. The kinases are: PKR-like kinase (PERK). PERK activation sends both pro-and anti-apoptotic signals but its main function is translation attenuation via phosphorylation of eIF2α which reduces the ER load. ATF6 induces expression of chaperones like BiP, but also the apoptosis factor CHOP. IRE1 is activated and becomes an endonuclease that process the XBP1 mRNA. XBP1 protein is a transcription factor that drives the transcription of both pro- and anti-apoptotic genes. The delicate balance between the protective and destructive branches of the UPR determines if the cell will overcome the stress or will die via the PCD pathway.

Figure 3

The mechanism of SLS. In trypanosomes all mRNA aretrans-spliced. In this process, the exon or spliced leader is donated to the mRNA from a small RNA, the spliced leader RNA. The SL RNA is transcribed and assembled in a distinct nuclear site that was termed the SL factory [5], where the SL RNA is transcribed, modified and assembles with its binding protein. Under stress that perturbs the ER homeostasis such as blocking translocation of proteins across the ER membrane (via RNAi silencing of SRP receptor, SEC61, or SEC63) or by prolonged exposure to chemicals such as DTT and 2DG or under drastic pH changes, the SLS pathway is induced. The hallmarks of SLS are shut-off in SL RNA transcription due to the inability of tSNA42 to bind to the SL RNA promoter, leading to the accumulation of the tSNAP42 in the nucleus. The shut-off of SL RNA transcription leads to marked reduction in mRNA production and to induction of apoptosis. One key kinase in this pathway is PK-3, a serine-threonine kinase that transmits the signal from the ER to the nucleus. Despite the fact that trans-splicing is inhibited during SLS, the level of certain mRNAs is increased. These mRNAs may lead the synthesis of proteins that are essential for executing SLS.