Like other Kinetoplastids, gene expression in Leishmania species is overwhelmingly post-transcriptionally controlled. This elevates the importance of RNA binding proteins (RBPs) in these systems as the primary gene regulators. Building upon the L. mexicana RBPome we isolated previously from the 3 main parasite lifecycle stages (Pablos et al. MCP, 2019), 70 non-basal RBPs were selected toward further investigation. An L. mexicana barcoded trans-regulator knockout clone library was created using CRISPR-cas9 (Baker et al. Nat Comms, 2021) and screened through lifecycle progression and macrophage or mouse infections.
Remarkably, 60% of the RBPs screened are essential for cell viability and 26% contribute to lifecycle progression to human-infectious stages, infectivity and/or virulence. Examination of individual knockout lines verify the screen outcomes of specific RBPs essential for parasite growth, viability and infectivity. 13 RBPs were endogenously tagged, immunoprecipitated and submitted for transcriptomic and proteomic analyses to identify all RNP components. Discrete complexes have been identified that may represent novel virulence factors. Further analyses are underway to map interaction dynamics of these key RNP regulators that drive differentiation and virulence capacity in Leishmania.
We present the first quantitative RBPome and Whole Cell proteome comparison between the 3 main lifecycle stages of Trypanosoma cruzi parasites. Lifecycle progression and bespoke adaptation to distinct host environments demand precise gene expression that is predominantly dependent upon post-transcriptional control. Constitutive transcription elevates the importance of RNA binding proteins for gene regulation in these parasites, yet strikingly few T.cruzi trans-regulators are characterized relative to other kinetoplastids. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of the stage-specific RBPomes and whole cell proteomes of the main T.cruzi lifecycle stages. Remarkably, while the whole cell proteomes display characteristically distinct surface proteins between these lifecycle stages as expected, the RBPomes of the human-infectious trypomastigote and amastigote stages bear striking similarities.
The presence of basal translational machinery and expected markers and near-identical proteomes derived from crosslinked and non-crosslinked cells in all sample sets support the validity of these proteomes. These represent the most in depth T.cruzi proteomes to date with outstanding coverage. Comparisons between these datasets provides unique insight into key candidate regulators that may prove essential to parasite survival, longevity and virulence.These proteomes can further enable in depth comparisons between Kinetoplastid species toward common essential regulators and host-adaptative mechanisms. Outco
