Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion

Baron, Nofar; Tupperwar, Nitin; Dahan, Irit; Hadad, Uzi; Davidov, Geula; Zarivach, Raz; Shapira, Michael Y.

doi:10.1371/journal.pntd.0008352

Cited by 9 publications

(25 citation statements)

References 58 publications

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“…We found clear evidence for association of 4EIP with the terminal uridylyl transferase TUT3, although its role is uncertain, and cells lacking 4EIP also had decreased levels of EIF4E1 protein. The protein 4EIP2 was associated with trypanosome EIF4E1 independently of 4EIP, suggesting binding to EIF4E1 as already indicated for Leishmania [28]. Both N-and C-terminally tagged versions of T. brucei 4EIP2 are in the cytoplasm [53]; high-throughput RNAi detected no defect for 4EIP2 depletion [49], but the degree of depletion was of course not assessed.…”

Section: Discussionmentioning

confidence: 86%

“…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 [ 27 ]. EIF4Es 3–6 have EIF4G partners and are probably all active in translation initiation, while the role of EIF4E2 is unclear [ 28 , 29 ]. Much of the evidence concerning EIF4E1 comes from Leishmania , which grow intracellularly as amastigotes within vertebrates, and as promastigotes within the sandfly vector.…”

Section: Introductionmentioning

confidence: 99%

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The EIF4E1-4EIP cap-binding complex of Trypanosoma brucei interacts with the terminal uridylyl transferase TUT3

2021

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Most transcription in Trypanosoma brucei is constitutive and polycistronic. Consequently, the parasite relies on post-transcriptional mechanisms, especially affecting translation initiation and mRNA decay, to control gene expression both at steady-state and for adaptation to different environments. The parasite has six isoforms of the cap-binding protein EIF4E as well as five EIF4Gs. EIF4E1 does not bind to any EIF4G, instead being associated with a 4E-binding protein, 4EIP. 4EIP represses translation and reduces the stability of a reporter mRNA when artificially tethered to the 3’-UTR, whether or not EIF4E1 is present. 4EIP is essential during the transition from the mammalian bloodstream form to the procyclic form that lives in the Tsetse vector. In contrast, EIF4E1 is dispensable during differentiation, but is required for establishment of growing procyclic forms. In Leishmania, there is some evidence that EIF4E1 might be active in translation initiation, via direct recruitment of EIF3. However in T. brucei, EIF4E1 showed no detectable association with other translation initiation factors, even in the complete absence of 4EIP. There was some evidence for interactions with NOT complex components, but if these occur they must be weak and transient. We found that EIF4E1is less abundant in the absence of 4EIP, and RNA pull-down results suggested this might occur through co-translational complex assembly. We also report that 4EIP directly recruits the cytosolic terminal uridylyl transferase TUT3 to EIF4E1/4EIP complexes. There was, however, no evidence that TUT3 is essential for 4EIP function.

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Section: Discussionmentioning

confidence: 86%

Section: Introductionmentioning

confidence: 99%

The EIF4E1-4EIP cap-binding complex of Trypanosoma brucei interacts with the terminal uridylyl transferase TUT3

2021

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“…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.…”

Section: Discussionmentioning

confidence: 90%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Falk

Marucha

Clayton

2021

Preprint

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Most transcription in Trypanosoma brucei is constitutive and polycistronic. Consequently, the parasite relies on post-transcriptional mechanisms, especially affecting translation initiation and mRNA decay, to control gene expression both at steady-state and for adaptation to different environments. The parasite has six isoforms of the cap-binding protein EIF4E as well as five EIF4Gs. EIF4E1 does not bind to any EIF4G, instead being associated with a 4E-binding protein, 4EIP. 4EIP represses translation and reduces the stability of a reporter mRNA when artificially tethered to the 3'-UTR, whether or not EIF4E1 is present. 4EIP is essential during the transition from the mammalian bloodstream form to the procyclic form that lives in the Tsetse vector. In contrast, EIF4E1 is dispensable during differentiation, but is required for establishment of growing procyclic forms. There are two competing models for EIF4E1 function: either EIF4E1 has translation initiation activity that is inhibited by 4EIP, or EIF4E1 acts only as an inhibitor. We here provide evidence for the second hypothesis. Even in the complete absence of 4EIP, EIF4E1 showed no detectable association with other translation initiation factors, and 4EIP loss caused no detectable change in 4E1-associated mRNAs. We found that 4EIP stabilises EIF4E1, probably through co-translational complex assembly, and that 4EIP directly recruits the cytosolic terminal uridylyl transferase TUT3 to EIF4E1/4EIP complexes. There was, however, no evidence that TUT3 is essential for 4EIP function; instead, some evidence implicated the NOT deadenylase complex.

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Shrivastava

Tupperwar

Schwartz

et al. 2021

IJMS

Self Cite

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Leishmania parasites cycle between sand fly vectors and mammalian hosts, transforming from extracellular promastigotes that reside in the vectors’ alimentary canal to obligatory intracellular non-motile amastigotes that are harbored by macrophages of the mammalian hosts. The transition between vector and host exposes them to a broad range of environmental conditions that induces a developmental program of gene expression, with translation regulation playing a key role. The Leishmania genome encodes six paralogs of the cap-binding protein eIF4E. All six isoforms show a relatively low degree of conservation with eIF4Es of other eukaryotes, as well as among themselves. This variability could suggest that they have been assigned discrete roles that could contribute to their survival under the changing environmental conditions. Here, we describe LeishIF4E-5, a LeishIF4E paralog. Despite the low sequence conservation observed between LeishIF4E-5 and other LeishIF4Es, the three aromatic residues in its cap-binding pocket are conserved, in accordance with its cap-binding activity. However, the cap-binding activity of LeishIF4E-5 is restricted to the promastigote life form and not observed in amastigotes. The overexpression of LeishIF4E-5 shows a decline in cell proliferation and an overall reduction in global translation. Immuno-cytochemical analysis shows that LeishIF4E-5 is localized in the cytoplasm, with a non-uniform distribution. Mass spectrometry analysis of proteins that co-purify with LeishIF4E-5 highlighted proteins involved in RNA metabolism, along with two LeishIF4G paralogs, LeishIF4G-1 and LeishIF4G-2. These vary in their conserved eIF4E binding motif, possibly suggesting that they can form different complexes.

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Distinct features of the Leishmania cap-binding protein LeishIF4E2 revealed by CRISPR-Cas9 mediated hemizygous deletion

Cited by 9 publications

References 58 publications

The EIF4E1-4EIP cap-binding complex of Trypanosoma brucei interacts with the terminal uridylyl transferase TUT3

The EIF4E1-4EIP cap-binding complex of Trypanosoma brucei interacts with the terminal uridylyl transferase TUT3

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

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

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