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

ABSTRACTIn 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.

Phylogenic analysis of Chinese Leishmania isolates based on small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA)

The leishmaniases are zoonotic diseases caused by protozoan parasites of the genus Leishmania. Leishmaniases are still endemic in China, especially in the west and northwest froniter regions. To revalue the preliminary phylogenetic results of Chinese Leishmania isolates, we amplified partial fragment of small subunit ribosomal RNA (SSU rRNA) and 7 spliced leader RNA (7SL RNA), then tested the phylogenetic relationships among Chinese Leishmania isolates and their relatives by analyzing SSU rRNA gene sequences and 7SL RNA gene sequences. 19 SSU RNA sequences and 9 7SL RNA sequences were obtained in our study, then analyzed with 42 SSU RNA sequences and 32 7SL RNA sequences retrieved from Genbank, respectively. In the Bayesian analysis of the SSU RNA gene, the isolate MHOM/CN/93/GS7 and the isolate IPHL/CN/77/XJ771 are members of Leishmania donovani complex, while the isolate MHOM/CN/84/JS1 clustered with Leishmania tropica. The other 11 Chinese Leishmania isolates (MHOM/CN/90/WC, MCAN/CN/90/SC11, MHOM/CN/80/XJ801, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/ 89/GS5) form an unclassified group, defined as Leishmania sp., and the most relative species to this group is L. tarentolae. In the Bayesian analysis of the 7SL RNA gene, 9 Chinese Leishmania isolates also formed an unclassified group with L. tarentolae, including canine isolate 10, MHOM/CN/85/GS4, MHOM/CN/84/SD1, MCAN/CN/86/SC7, MHOM/CN/54/#3, MHOM/ CN/83/GS2, MHOM/CN/90/SC10H2, MHOM/CN/89/GS6 and MHOM/CN/89/GS5. We concluded that: (1) Chinese Leishmania isolates are non-monophyly group; (2) an unclassified group may exist in China, and the most relative species to this group is L. tarentolae; (3) MHOM/CN/84/JS1, which was previously assigned as L. donovani, was most genetically related to L. tropica strain MHOM/SU/74/K27.