Phosphorylation of the TATA-binding protein activates the spliced leader silencing pathway in Trypanosoma brucei

Abstract

The parasite Trypanosoma brucei is the causative agent of human African sleeping sickness. T. brucei genes are constitutively transcribed in polycistronic units that are processed by trans-splicing and polyadenylation. All mRNAs are trans-spliced to generate mRNAs with a common 5' exon derived from the spliced leader RNA (SL RNA). Persistent endoplasmic reticulum (ER) stress induces the spliced leader silencing (SLS) pathway, which inhibits trans-splicing by silencing SL RNA transcription, and correlates with increased programmed cell death. We found that during ER stress induced by SEC63 silencing or low pH, the serine-threonine kinase PK3 translocated from the ER to the nucleus, where it phosphorylated the TATA-binding protein TRF4, leading to the dissociation of the transcription preinitiation complex from the promoter of the SL RNA encoding gene. PK3 loss of function attenuated programmed cell death induced by ER stress, suggesting that SLS may contribute to the activation of programmed cell death.

Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

“…From the outset it must be noted that pharmacological and genetic characterization of SLS as a UPR-like response has been done primarily in PCF trypanosomes (4,7,8,11), and this may contribute to some of the discrepancies with the work we present here, which is performed exclusively in BSF parasites. Nevertheless, two of the central stimuli used to elucidate these responses in PCF cells, disruption of the translocation machinery and DTT treatment, were applied to BSF trypanosomes with similar results, and this has been used to argue that SLS is not a stage-specific phenomenon (4).…”

“…In light of these results, it is difficult to see how ablating the translocation machinery constitutes a legitimate internal ER stress. Thus, the only connection between persistent ER stress and the SLS response is the claim that DTT treatment has the same effect as RNAi targeting the aforementioned translocation factors (4,7,8,11). Our work here disqualifies DTT, and in our opinion, as with the UPR-like response, any conclusions about SL silencing based on use of this reagent should be regarded with extreme caution.…”

“…SLS correlates with upregulation and mislocalization within the nucleus of tSNAP42 (also known as SNAP2), a key transcription factor for SL RNA synthesis (9,10), and its release from the SL promoter. More recently, a signaling pathway leading from a putative ER-associated kinase (TbeIFK3) to the SL transcription preinitiation complex has been described (11). Most of this genetic and molecular work was performed in procyclic culture forms (PCF), corresponding to insect midgut parasites, but loss of SL RNA was also observed in mammalian bloodstreamform (BSF) cells upon TbSec63 knockdown (4).…”

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

“…A). Since SNAP2 remained associated with the SL RNA transcription complex, in SEC63 / SKCRP7.1/7.2 silenced cells, unlike in SEC63 silenced cells (Hope et al ., ), SKCRP7.1/7 .2 may function as part of the steps between PK3 activation and TRF4 phosphorylation in the nucleus.…”

“…We have recently shown that a serine/threonine kinase known as TbeIF3 (or termed by us, PK3), translocates from the ER to the nucleus where it phosphorylates the TRF4 or the TATA‐binding protein, leading to its dissociation from the SL RNA promoter. In SEC63 / PK3 co‐silenced cells, SLS is not induced, and apoptosis is compromised (Hope et al ., ). Our results (Fig.…”