Dataset Information

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei.

ABSTRACT: Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD-001 selection (RIT-seq). The resistance phenotypes were confirmed by individual RNAi knockdown. Highest level of resistance to NPD-001, approximately 17-fold, was seen for knockdown of CARP7 (Tb927.7.4510). CARP1 and CARP11 contain predicted cyclic AMP binding domains and bind cAMP as evidenced by capture and competition on immobilised cAMP. CARP orthologues are strongly enriched in kinetoplastid species, and CARP3 and CARP11 are unique to Trypanosoma. Localization data and/or domain architecture of all CARPs predict association with the T. brucei flagellum. This suggests a crucial role of cAMP in flagellar function, in line with the cell division phenotype caused by high cAMP and the known role of the flagellum for cytokinesis. The CARP collection is a resource for discovery of unusual cAMP pathways and flagellar biology.

SUBMITTER: Bachmaier S

PROVIDER: S-EPMC10354285 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in <i>Trypanosoma brucei</i>.

Bachmaier Sabine S Gould Matthew K MK Polatoglou Eleni E Omelianczyk Radoslaw R Brennand Ana E AE Aloraini Maha A MA Munday Jane C JC Horn David D Boshart Michael M de Koning Harry P HP

Frontiers in cellular and infection microbiology 20230705

Cyclic AMP signalling in trypanosomes differs from most eukaryotes due to absence of known cAMP effectors and cAMP independence of PKA. We have previously identified four genes from a genome-wide RNAi screen for resistance to the cAMP phosphodiesterase (PDE) inhibitor NPD-001. The genes were named cAMP Response Protein (CARP) 1 through 4. Here, we report an additional six CARP candidate genes from the original sample, after deep sequencing of the RNA interference target pool retrieved after NPD- ...[more]

PMID: 37475965

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Project description:Eukaryotic ribosome biogenesis is an essential cellular process involving tightly coordinated assembly of multiple rRNA and protein components. Much of our understanding of this pathway has come from studies performed with yeast model systems. These studies have identified critical checkpoints in the maturation of the large ribosomal subunit (LSU/60S), one of which is the proper formation and incorporation of the 5S ribonucleoprotein complex (5S RNP). Research on the 5S RNP has identified a complex containing the four proteins L5, L11, Rpf2, and Rrs1 as well as 5S rRNA. Our laboratory has studied the 5S RNP in Trypanosoma brucei, a eukaryotic parasite, and identified the proteins P34 and P37 as essential, parasite-specific members of this complex. We have additionally identified homologues of L5, Rpf2, Rrs1, and 5S rRNA in T. brucei and characterized their roles in this essential process. In this study, we examined the T. brucei homologue of ribosomal protein L11 as a member of the 5S RNP. We showed that TbL11 is essential and that it is important for proper ribosome subunit formation and 60S rRNA processing. Additionally, we identified TbL11 interactions with TbL5 and TbRpf2, as well as novel interactions with the kinetoplast-specific proteins P34 and P37. These findings expand our understanding of a crucial process outside the context of model yeast organisms and highlight differences in an otherwise highly conserved process that could be used to develop future treatments against T. bruceiIMPORTANCE The human-pathogenic, eukaryotic parasite Trypanosoma brucei causes human and animal African trypanosomiases. Treatments for T. brucei suffer from numerous hurdles, including adverse side effects and developing resistance. Ribosome biogenesis is one critical process for T. brucei survival that could be targeted for new drug development. A critical checkpoint in ribosome biogenesis is formation of the 5S RNP, which we have shown involves the trypanosome-specific proteins P34 and P37 as well as homologues of Rpf2, Rrs1, and L5. We have identified parasite-specific characteristics of these proteins and involvement in key parts of ribosome biogenesis, making them candidates for future drug development. In this work, we characterized the T. brucei homologue of ribosomal protein L11. We show that it is essential for parasite survival and is involved in ribosome biogenesis and rRNA processing. Furthermore, we identified novel interactions with P34 and P37, characteristics that make this protein a potential target for novel chemotherapeutics.

Dataset Information

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in Trypanosoma brucei.

Publications

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance in <i>Trypanosoma brucei</i>.

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