Phosphorylation and interactions associated with the control of the<i>Leishmania</i>Poly-A Binding Protein 1 (PABP1) function during translation initiation

Neto, Osvaldo P. de Melo; Lima, Tamara de Carli da Costa; Merlo, Kleison C.; Romão, Tatiany Patrícia; Rocha, Pollyanna O.; Assis, Ludmila A.; Nascimento, Larissa Mélo do; Xavier, Camila Cavalcanti; Rezende, Antônio Mauro; Reis, Christian Robson de Souza; Papadopoulou, Barbara

doi:10.1080/15476286.2018.1445958

Cited by 11 publications

(33 citation statements)

References 82 publications

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“…For Leishmania eIF4E4, additional interaction with PABP2 was shown [ 35 ]. Moreover, while this manuscript was in revision, a PABP1 interactome for Leishmania infantum was published [ 62 ] and is in agreement with our data: seven proteins consistently co-precipitated with Leishmania PABP1, of which six correspond to the six most enriched proteins in the T . brucei PABP1 pulldown (eIF4E4, eIF4G3, PABP1, RBP23, Tb927.7.7460, ZC3H41) and only one protein (Tb927.10.13800) was not identified with our conditions.…”

Section: Resultssupporting

confidence: 88%

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

et al. 2018

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Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.

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Section: Resultssupporting

confidence: 88%

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

et al. 2018

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“…This finding is consistent with the results of previous (qualitative) pull-down and two-hybrid assay experiments performed with PABP1 and eIF4E4 (7). Another report has attempted to predict molecular interactions between Leishmania PABP1 and eIF4E4 by modeling MLLE and PAM2 domains from other (human) proteins (33). In order to obtain definitive experimental information on the interaction architecture of the Leishmania proteins, we decided to perform structural analysis on the PABP1 and eIF4E4 domains identified by our interaction screening procedure.…”

Section: Resultsmentioning

confidence: 99%

TheLeishmaniaPABP1–eIF4E4 interface: a novel 5′–3′ interaction architecture for trans-spliced mRNAs

Rodrigues

Firczuk

Breeze

et al. 2018

Nucleic Acids Research

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Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5′ cap4 structure (m7Gpppm36,6,2′Apm2′Apm2′Cpm23,2′U). NMR and X-ray crystallography reveal that Leishmania has a unique type of N-terminally-extended cap-binding protein (eIF4E4) that binds via a PAM2 motif to PABP1. This relies on the interactions of a combination of polar and charged amino acid side-chains together with multiple hydrophobic interactions, and underpins a novel architecture in the Leishmania cap4-binding translation factor complex. Measurements using microscale thermophoresis, fluorescence anisotropy and surface plasmon resonance characterize the key interactions driving assembly of the Leishmania translation initiation complex. We demonstrate that this complex can accommodate Leishmania eIF4G3 which, unlike the standard eukaryotic initiation complex paradigm, binds tightly to eIF4E4, but not to PABP1. Thus, in Leishmania, the chain of interactions 5′cap4-eIF4E4–PABP1-poly(A) bridges the mRNA 5′ and 3′ ends. Exceptionally, therefore, by binding tightly to two protein ligands and to the mRNA 5′ cap4 structure, the trypanosomatid N-terminally extended form of eIF4E acts as the core molecular scaffold for the mRNA-cap-binding complex. Finally, the eIF4E4 N-terminal extension is an intrinsically disordered region that transitions to a partly folded form upon binding to PABP1, whereby this interaction is not modulated by poly(A) binding to PABP1.

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“…The six Leishmania cap-binding protein paralogs vary from each other in their sequences, affinities to the mRNA cap structures, and binding partners. These variations suggest that each LeishIF4E paralog is responsible for different functions, although overlaps are also expected (15, 28). This study aimed to investigate the role of LeishIF4E1 in Leishmania by testing how its deletion affected the proteomic profile of the deletion mutant and the subsequent effect on broad aspects of parasite morphology, metabolism, growth, and virulence.…”

Section: Discussionmentioning

confidence: 96%

LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity

Tupperwar

Shrivastava

Shapira

2019

mSphere

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Leishmania parasites are the causative agents of a broad spectrum of diseases. The parasites migrate between sand-fly vectors and mammalian hosts, adapting to changing environments by driving a regulated program of gene expression, with translation regulation playing a key role. The leishmanias encode six different paralogs of eIF4E, the cap-binding translation initiation factor. Since these vary in function, expression profile, and assemblage, it is assumed that each is assigned a specific role throughout the life cycle. Using the CRISPR-Cas9 system for Leishmania, we generated a null mutant of LeishIF4E1, eliminating both alleles. Although the mutant cells were viable, their morphology was altered and their ability to synthesize the flagellum was impaired. Elimination of LeishIF4E1 affected their protein expression profile and decreased their ability to infect cultured macrophages. Restoring LeishIF4E1 expression restored the affected features. This study highlights the importance of LeishIF4E1 in diverse cellular events during the life cycle of Leishmania.

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Phosphorylation and interactions associated with the control of theLeishmaniaPoly-A Binding Protein 1 (PABP1) function during translation initiation

Cited by 11 publications

References 82 publications

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

TheLeishmaniaPABP1–eIF4E4 interface: a novel 5′–3′ interaction architecture for trans-spliced mRNAs

LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity

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