RT Journal Article SR Electronic T1 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 JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.09.07.459367 DO 10.1101/2021.09.07.459367 A1 Okalang, Uthman A1 Bar-Ner, Bar Mualem A1 Rajan, K. Shanmugha A1 Friedman, Nehemya A1 Aryal, Saurav A1 Egarmina, Katarina A1 Hope, Ronen A1 Khazanov, Netaly A1 Senderowitz, Hanoch A1 Alon, Assaf A1 Fass, Deborah A1 Michaeli, Shulamit YR 2021 UL http://biorxiv.org/content/early/2021/09/08/2021.09.07.459367.abstract AB In the parasite Trypanosoma brucei, the causative agent of human African sleeping sickness, all mRNAs are trans-spliced to generate a common 5’ exon derived from the spliced leader RNA (SL RNA). Perturbations of protein translocation across the endoplasmic reticulum (ER) induce the spliced leader RNA silencing (SLS) pathway. SLS activation is mediated by a serine-threonine kinase, PK3, which translocates from the cytosolic face of the ER to the nucleus, where it phosphorylates the TATA binding protein TRF4, leading to the shut-off of SL RNA transcription, followed by induction of programmed cell death. Here, we demonstrate that SLS is also induced by depletion of the essential ER resident chaperones BiP and calreticulin, ER oxidoreductin 1 (ERO1), and the Golgi-localized quiescin sulfhydryl oxidase (QSOX1). Most strikingly, silencing of Rhomboid-like 1(TIMRHOM1) involved in mitochondrial protein import, also induces SLS. The PK3 kinase, which integrates SLS signals, is modified by phosphorylation on multiple sites. To determine which of the phosphorylation events activate PK3, several individual mutations or their combination were generated. These mutations failed to completely eliminate the phosphorylation or translocation of the kinase to the nucleus. The structure of PK3 kinase and its ATP binding domain were therefore modeled. A conserved phenylalanine at position 771 was proposed to interact with ATP, and the PK3F771L mutation completely eliminated phosphorylation under SLS, suggesting that the activation involves most if not all the phosphorylation sites. The study suggests that the SLS occurs broadly in response to failures in protein sorting, folding, or modification across multiple compartments.