Distinct features of theLeishmaniacap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated heterozygous deletion

Abstract

20 Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to 21 alternating environments by stage-differentiation, accompanied by changes in the proteome 22 profiles. Translation regulation plays a central role in driving the differential program of gene 23 expression, since control of gene regulation in Leishmania is mostly post-transcriptional. 24 The Leishmania genome encodes six eIF4E candidates, each assumed to have specific functions, 25 although overlaps are expected. It is noted that some of them can bind to a dedicated eIF4G 26 candidate partners, and LeishIF4E2 does not bind any known eIF4G ortholog. LeishIF4E2 was 27 previously shown to comigrate in the polysomal fractions of sucrose gradients, whereas initiation 28 factors usually comigrate with pre-initiation and initiation complexes. Using the CRISPR-Cas9 29 methodology, we deleted one of the two LeishIF4E2 gene copies. The deletion caused severe 30 alterations in the morphology of mutant cells that turned round and equipped with a very short 31 flagellum, but their growth rate and general translation remained unaffected. Proteomic analysis 32 of the LeishIF4E2(+/-) mutant cells compared to wild type controls showed that the number of 33 proteins that were upregulated exceeded the number of downregulated proteins, possibly 34 indicating that a repressor function was eliminated. The upregulated proteins were related mainly 35 to general metabolic processes, DNA repair and replication, signaling, and cellular motor 36 activity. The downregulated proteins included several groups, including cytoskeletal and 37 ribosomal proteins. Despite the fact that only one of the two LeishIF4E2 alleles was deleted, the 38 mutant cells were impaired in their ability to infect cultured macrophages. LeishIF4E2 does not 39 behave like a general translation factor and its function remains elusive. Although it could have a 40 repressive function, we cannot exclude the possibility that it is responsible for translation of a 41 specific set of transcripts. Overall, our results are in line with the possibility that the different 42 LeishIF4Es are assigned unique functions. 43 44 Author summary 45 Leishmania parasites cause a broad spectrum of diseases that lead to different 46 pathological symptoms. During their life cycle, the parasites shuffle between sand-fly 47 vectors and mammalian hosts, while adapting to changing environments via a stage 48 specific program of gene expression that assists the survival of Leishmania under the 49 changing conditions. Translation initiation plays a key role in control of gene expression, 50 in Leishmania this is exemplified by the presence of multiple cap-binding complexes that 51 interact with mRNAs. The parasites encode multiple paralogs of the cap-binding 52 translation initiation factor eIF4E, and of its corresponding binding partner eIF4G, 53 forming complexes with different potential functions. Using the CRISPR-Cas9 54 methodology, we generated a heterozygous mutant of the least studied cap-binding 55 pa...

Mechanisms of translation repression by the EIF4E1-4EIP cap-binding complex of Trypanosoma brucei: potential roles of the NOT complex and a terminal uridylyl transferase

“…The protein 4EIP2 was associated with trypanosome EIF4E1 independently of 4EIP, suggesting binding to EIF4E1 as already indicated for Leishmania (Baron et al 2021). Both N-and C-terminally tagged versions of T. brucei 4EIP2 are in the cytoplasm (Dean et al 2017); high-throughput RNAi detected no defect for 4EIP2 depletion (Alsford et al 2011), but the degree of depletion was of course not assessed.…”

“…In accordance with their need for adaptive translation regulation, kinetoplastid parasites are equipped with six eIF4E isoforms (EIF4E1-6), as well as five eIF4Gs (EIF4G1-5), the individual roles of which have been the subject of numerous studies (Freire et al 2017). EIF4Es 3-6 have EIF4G partners and are probably all active in translation initiation, while the role of EIF4E2 is unclear (Freire et al 2018; Baron et al 2021). Much of the evidence concerning EIF4E1 comes from Leishmania , which grow intracellularly as amastigotes within vertebrates, and as promastigotes within the sandfly vector.…”

Finding Correlations Between mRNA and Protein Levels in Leishmania Development: Is There a Discrepancy?

“…In the cytosol, the binding of eukaryotic translational initiation factors recruits ribosomes and initiates translation (Clayton, 2019) (Figure 3). Knockout of different translation initiation factors in Leishmania impacts the cells' whole proteome, affecting parasite morphology and infectivity (Tupperwar et al, 2019;Baron et al, 2020;Shrivastava et al, 2021). Posttranscriptional and posttranslational controls, including mRNA stability, FIGURE 2 | Graph showing the number of publications retrieved in PubMed using the terms "mRNA AND leishmania" (light blue bars), "protein AND leishmania" (red bars), "transcriptome AND leishmania" (dark blue bars), "proteome AND leishmania" (pink bars), "transcriptomic AND leishmania" (gray bars), and "proteomic AND leishmania" (purple bars).…”

“…In the cytosol, the binding of eukaryotic translational initiation factors recruits ribosomes and initiates translation ( Clayton, 2019 ) ( Figure 3 ). Knockout of different translation initiation factors in Leishmania impacts the cells’ whole proteome, affecting parasite morphology and infectivity ( Tupperwar et al., 2019 ; Baron et al., 2020 ; Shrivastava et al., 2021 ). Posttranscriptional and posttranslational controls, including mRNA stability, translation initiation and protein folding are mechanisms of gene expression regulation in Leishmania , indicating how complex the process from gene to protein can be ( Clayton and Shapira, 2007 ; Haile and Papadopoulou, 2007 ; Stiles et al., 2016 ).…”

LeishIF4E-5 Is a Promastigote-Specific Cap-Binding Protein in Leishmania

“…It does not appear to bind any of the eIF4G candidates and was found to migrate with the heavy polysomal complexes in sucrose gradients, thus could be associated with the stabilization of polysomes in Leishmania . Genome-wide tethering assay in Trypanosoma brucei classified LeishIF4E-2 as a putative translation repressor [ 18 ], but in Leishmania its role still remains elusive [ 19 ].…”