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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

Uthman Okalang, Bar Mualem Bar-Ner, K. Shanmugha Rajan, Nehemya Friedman, Saurav Aryal, Katarina Egarmina, Ronen Hope, Netaly Khazanov, Hanoch Senderowitz, Assaf Alon, Deborah Fass, View ORCID ProfileShulamit Michaeli
doi: https://doi.org/10.1101/2021.09.07.459367
Uthman Okalang
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Bar Mualem Bar-Ner
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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K. Shanmugha Rajan
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Nehemya Friedman
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Saurav Aryal
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Katarina Egarmina
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Ronen Hope
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Netaly Khazanov
2Chemistry Department, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Hanoch Senderowitz
2Chemistry Department, Bar-Ilan University, Ramat-Gan 52900, Israel.
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Assaf Alon
3Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
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Deborah Fass
3Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001, Israel
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Shulamit Michaeli
1The Mina and Everard Goodman Faculty of Life Sciences and Advanced Materials and Nanotechnology Institute, Bar-Ilan University, Ramat-Gan 52900, Israel.
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  • ORCID record for Shulamit Michaeli
  • For correspondence: Shulamit.Michaeli@biu.ac.il
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ABSTRACT

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.

Footnotes

  • ↵* The first three authors contributed equally to this study

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Posted September 08, 2021.
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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
Uthman Okalang, Bar Mualem Bar-Ner, K. Shanmugha Rajan, Nehemya Friedman, Saurav Aryal, Katarina Egarmina, Ronen Hope, Netaly Khazanov, Hanoch Senderowitz, Assaf Alon, Deborah Fass, Shulamit Michaeli
bioRxiv 2021.09.07.459367; doi: https://doi.org/10.1101/2021.09.07.459367
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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
Uthman Okalang, Bar Mualem Bar-Ner, K. Shanmugha Rajan, Nehemya Friedman, Saurav Aryal, Katarina Egarmina, Ronen Hope, Netaly Khazanov, Hanoch Senderowitz, Assaf Alon, Deborah Fass, Shulamit Michaeli
bioRxiv 2021.09.07.459367; doi: https://doi.org/10.1101/2021.09.07.459367

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