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Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System
Published on: July 31, 2019
Depletion of trypanosome CTR9 leads to gene expression defects
Benard A Ouna1, Benson Nyambega, Theresa Manful
1Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ/ZMBH Alliance, Heidelberg, Germany.
Abstract:
The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1.
Insights
The Paf1 complex subunit CTR9 is essential for trypanosome survival. Loss of CTR9 severely impacts essential mRNA levels and chromatin silencing, revealing its critical role in gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Parasitology
Background:
- The Paf1 complex is crucial for transcription and mRNA processing in eukaryotes.
- Its subunits are involved in histone modification and RNA polymerase activity.
- Trypanosome CTR9 is essential, but its specific role and interacting partners are unknown.
Purpose of the Study:
- To investigate the function and interacting partners of the essential trypanosome CTR9 protein.
- To understand the impact of CTR9 loss on gene expression and chromatin regulation in trypanosomes.
Main Methods:
- Tandem affinity purification to identify CTR9-interacting proteins.
- RNA interference (RNAi) to deplete CTR9 expression.
- Analysis of essential mRNA levels and chromatin-silenced gene expression.
Main Results:
- Trypanosome CTR9 interacts with homologues of Leo1 and Cdc73, but not Rtf1 or Paf1.
- RNAi-mediated depletion of CTR9 caused a >10-fold decrease in 131 essential mRNAs.
- Affected mRNAs included those critical for RNA polymerase function, mRNA processing, and RNA binding.
- Loss of CTR9 led to derepression of genes in silenced chromatin regions.
Conclusions:
- Trypanosome CTR9 is a vital component of a Paf1 complex-like machinery essential for trypanosome survival.
- CTR9 plays a critical role in maintaining the expression of essential genes and regulating chromatin silencing.
- These findings highlight CTR9's importance in trypanosome gene expression and its potential as a drug target.
