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

Abstract

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.

Exosome secretion affects social motility in Trypanosoma brucei

“…Inspecting TEM sections of SEC63 silenced cells, which induce the spliced leader silencing (SLS) pathway [53], revealed the presence of another type of vesicle, double-membrane autophagosomes [54], but not MVBs filled with ILVs, as in SmD1 silenced cells (S6 Fig); thus MVBs seem to be specific to cells secreting massive amounts of exosomes. Small numbers of exosomes are secreted from SEC63 silenced cells (S7 Fig).…”

“…Note, that the proteome of SEC63 silenced cells shares changes which are common to SmD1 silenced cells, possibly because these two type of cells are inhibited in trans -splicing. However, changes that are specific to each of these perturbations were also identified [54]. The repelling activity of these two silenced cells clearly indicate that the repelling signal(s) is not specific to dying cells (Fig 11).…”

The spliced leader RNA silencing (SLS) pathway in Trypanosoma brucei is induced by perturbations of endoplasmic reticulum, Golgi, or mitochondrial proteins factors and functional analysis of SLS inducing kinase, PK3

“…The proteome analysis of cells in which SLS had been induced by silencing of sec63 indicated an increase in the level of TIMRHOM1 protein (27). To verify this elevation, the level of the protein was examined upon SLS induced by sec63 silencing, and a fourfold increase was observed (Fig.…”

“…Cells expressing the SEC63 silencing construct and PK3 PTP-tagged protein (27), uninduced or induced for 2.5 days, were grown to a density of 2×10 7 cells ml -1`. The purification protocol used was essentially as described previously (33) from 10 10 cells but included the addition of phosphatase inhibitors to the extract (10 mM NaF, 1 mM sodium orthovanadate and 50 mM beta-glycerophosphate).…”

Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part III: Protozoa (3): Kinetoplastids

“…Later, SKCRPs 7.1 and 7.2 were identified in T. brucei under persistent endoplasmic reticulum stress. The investigators concluded the essential role played by SKCRPs to induce PCD in under-stressed parasites [208] .…”