Transcriptome and proteome analyses and the role of atypical calpain protein and autophagy in the spliced leader silencing pathway in Trypanosoma brucei.

Under persistent ER stress, Trypanosoma brucei parasites induce the spliced leader silencing (SLS) pathway. In SLS, transcription of the SL RNA gene, the SL donor to all mRNAs, is extinguished, arresting trans-splicing and leading to programmed cell death (PCD). In this study, we investigated the transcriptome following silencing of SEC63, a factor essential for protein translocation across the ER membrane, and whose silencing induces SLS. The proteome of SEC63-silenced cells was analyzed with an emphasis on SLS-specific alterations in protein expression, and modifications that do not directly result from perturbations in trans-splicing. One such protein identified is an atypical calpain SKCRP7.1/7.2. Co-silencing of SKCRP7.1/7.2 and SEC63 eliminated SLS induction due its role in translocating the PK3 kinase. This kinase initiates SLS by migrating to the nucleus and phosphorylating TRF4 leading to shut-off of SL RNA transcription. Thus, SKCRP7.1 is involved in SLS signaling and the accompanying PCD. The role of autophagy in SLS was also investigated; eliminating autophagy through VPS34 or ATG7 silencing demonstrated that autophagy is not essential for SLS induction, but is associated with PCD. Thus, this study identified factors that are used by the parasite to cope with ER stress and to induce SLS and PCD.

Spliced-leader RNA silencing: a novel stress-induced mechanism in Trypanosoma brucei.

The signal-recognition particle (SRP) mediates the translocation of membrane and secretory proteins across the endoplasmic reticulum upon interaction with the SRP receptor. In trypanosomes, the main RNA molecule is the spliced-leader (SL) RNA, which donates the SL sequence to all messenger RNA through trans-splicing. Here, we show that RNA interference silencing of the SRP receptor (SRalpha) in Trypanosoma brucei caused the accumulation of SRP on ribosomes and triggered silencing of SL RNA (SLS). SLS was elicited due to the failure of the SL RNA-specific transcription factor tSNAP42 to bind to its promoter. SL RNA reduction, in turn, eliminated mRNA processing and resulted in a significant reduction of all mRNA tested. SLS was also induced under pH stress and might function as a master regulator in trypanosomes. SLS is reminiscent of, but distinct from, the unfolded protein response and can potentially act as a new target for parasite eradication.