The Leishmania PABP1-eIF4E4 interface: A novel 5′-3′ interaction architecture for trans-spliced mRNAs

Fabio Henrique Dos Santos Rodrigues; Helena Firczuk; Alexander L. Breeze; Alexander D. Cameron; Martin Walko; Andrew J. Wilson; Nilson I.T. Zanchin; John E.G. McCarthy

doi:10.1093/nar/gky1187

The Leishmania PABP1-eIF4E4 interface: A novel 5′-3′ interaction architecture for trans-spliced mRNAs

Fabio Henrique Dos Santos Rodrigues, Helena Firczuk, Alexander L. Breeze, Alexander D. Cameron, Martin Walko, Andrew J. Wilson, Nilson I.T. Zanchin, John E.G. McCarthy^*

^*Corresponding author for this work

Chemistry

Research output: Contribution to journal › Article › peer-review

8 Citations (Scopus)

Abstract

Trans-splicing of trypanosomatid polycistronic transcripts produces polyadenylated monocistronic mRNAs modified to form the 5′ cap4 structure (m⁷Gpppm₃^6,6,2′Apm^2′Apm^2′Cpm₂^3,2′U). NMR and Xray 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.

Original language	English
Pages (from-to)	1493-1504
Number of pages	12
Journal	Nucleic Acids Research
Volume	47
Issue number	3
DOIs	https://doi.org/10.1093/nar/gky1187
Publication status	Published - 20 Feb 2019

Bibliographical note

Funding Information:
Author contributions: J.E.G.M., F.H.dS.R., H.F., A.L.B., A.C., N.I.T.Z. and A.J.W. designed the research; F.H.dS.R. and H.F. performed the research; M.W. contributed peptides; J.E.G.M., A.L.B. and A.C. analyzed the data; J.E.G.M. wrote the paper with contributions from F.H.dS.R, A.C., A.L.B. and A.J.W. In the School of Life Sciences at the University of Warwick we thank Deborah Brotherton for advice on protein production and purification, Avinash Punekar for crystallographic data collection at the Diamond Light Source (proposal mx14692), as well as Cleidi Zampronio and Alex Jones for help with mass spectrometry. Protein production and biomolecular interaction studies via surface plasmon resonance, microscale thermophoresis and fluorescence anisotropy were performed using equipment in the WISB Research Technology Facility. We acknowledge access to the Astbury Biostructure Laboratory BioNMR Facility (University of Leeds) and also thank Arnout Kalverda (BioNMR Facility) for NMR technical assistance. Medical Research Council funding [MR/N017447/1] to the Universities of Warwick and Leeds; Fundação Araucária funding to the Instituto Carlos Chagas (FIOCRUZParaná) [005/2016]; Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the award of a Ciência sem Fronteiras (Science Without Borders) studentship to Fábio Henrique dos Santos Rodrigues. WISB is funded by the BBSRC and EPSRC [BB/M017982/1] and the University of Warwick. Funding to the Astbury Biostructure Laboratory BioNMR Facility from the Wellcome Trust [104920/Z/14/Z]; University of Leeds; EPSRC funding [EP/N035267/1 to M.W.]; peptide production, purification and characterization infrastructure supported by EPSRC [EP/N013573/1, EP/KO39292/1]; Wellcome Trust [WT097827/Z/11/A]. Funding for open access charge: University of Warwick.

Funding Information:
Medical Research Council funding [MR/N017447/1] to the Universities of Warwick and Leeds; Fundac¸ão Araucária funding to the Instituto Carlos Chagas (FIOCRUZ-Paraná) [005/2016]; Brazilian Conselho Nacional de De-senvolvimento Científico e Tecnológico (CNPq) for the award of a Ciência sem Fronteiras (Science Without Borders) studentship to Fábio Henrique dos Santos Ro-drigues. WISB is funded by the BBSRC and EPSRC [BB/M017982/1] and the University of Warwick. Funding to the Astbury Biostructure Laboratory BioNMR Facility from the Wellcome Trust [104920/Z/14/Z]; University of Leeds; EPSRC funding [EP/N035267/1 to M.W.]; peptide production, purification and characterization infrastructure supported by EPSRC [EP/N013573/1, EP/KO39292/1]; Wellcome Trust [WT097827/Z/11/A]. Funding for open access charge: University of Warwick. Conflict of interest statement. None declared.

Publisher Copyright:
© The Author(s) 2018.

ASJC Scopus subject areas

Genetics

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10.1093/nar/gky1187

Cite this

@article{a116d950dc0a4835b182dd49634f43b9,

title = "The Leishmania PABP1-eIF4E4 interface: A novel 5′-3′ interaction architecture for trans-spliced mRNAs",

abstract = "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 Xray 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.",

author = "{Dos Santos Rodrigues}, {Fabio Henrique} and Helena Firczuk and Breeze, {Alexander L.} and Cameron, {Alexander D.} and Martin Walko and Wilson, {Andrew J.} and Zanchin, {Nilson I.T.} and McCarthy, {John E.G.}",

note = "Funding Information: Author contributions: J.E.G.M., F.H.dS.R., H.F., A.L.B., A.C., N.I.T.Z. and A.J.W. designed the research; F.H.dS.R. and H.F. performed the research; M.W. contributed peptides; J.E.G.M., A.L.B. and A.C. analyzed the data; J.E.G.M. wrote the paper with contributions from F.H.dS.R, A.C., A.L.B. and A.J.W. In the School of Life Sciences at the University of Warwick we thank Deborah Brotherton for advice on protein production and purification, Avinash Punekar for crystallographic data collection at the Diamond Light Source (proposal mx14692), as well as Cleidi Zampronio and Alex Jones for help with mass spectrometry. Protein production and biomolecular interaction studies via surface plasmon resonance, microscale thermophoresis and fluorescence anisotropy were performed using equipment in the WISB Research Technology Facility. We acknowledge access to the Astbury Biostructure Laboratory BioNMR Facility (University of Leeds) and also thank Arnout Kalverda (BioNMR Facility) for NMR technical assistance. Medical Research Council funding [MR/N017447/1] to the Universities of Warwick and Leeds; Funda{\c c}{\~a}o Arauc{\'a}ria funding to the Instituto Carlos Chagas (FIOCRUZParan{\'a}) [005/2016]; Brazilian Conselho Nacional de Desenvolvimento Cient{\'i}fico e Tecnol{\'o}gico (CNPq) for the award of a Ci{\^e}ncia sem Fronteiras (Science Without Borders) studentship to F{\'a}bio Henrique dos Santos Rodrigues. WISB is funded by the BBSRC and EPSRC [BB/M017982/1] and the University of Warwick. Funding to the Astbury Biostructure Laboratory BioNMR Facility from the Wellcome Trust [104920/Z/14/Z]; University of Leeds; EPSRC funding [EP/N035267/1 to M.W.]; peptide production, purification and characterization infrastructure supported by EPSRC [EP/N013573/1, EP/KO39292/1]; Wellcome Trust [WT097827/Z/11/A]. Funding for open access charge: University of Warwick. Funding Information: Medical Research Council funding [MR/N017447/1] to the Universities of Warwick and Leeds; Fundac¸{\~a}o Arauc{\'a}ria funding to the Instituto Carlos Chagas (FIOCRUZ-Paran{\'a}) [005/2016]; Brazilian Conselho Nacional de De-senvolvimento Cient{\'i}fico e Tecnol{\'o}gico (CNPq) for the award of a Ci{\^e}ncia sem Fronteiras (Science Without Borders) studentship to F{\'a}bio Henrique dos Santos Ro-drigues. WISB is funded by the BBSRC and EPSRC [BB/M017982/1] and the University of Warwick. Funding to the Astbury Biostructure Laboratory BioNMR Facility from the Wellcome Trust [104920/Z/14/Z]; University of Leeds; EPSRC funding [EP/N035267/1 to M.W.]; peptide production, purification and characterization infrastructure supported by EPSRC [EP/N013573/1, EP/KO39292/1]; Wellcome Trust [WT097827/Z/11/A]. Funding for open access charge: University of Warwick. Conflict of interest statement. None declared. Publisher Copyright: {\textcopyright} The Author(s) 2018.",

year = "2019",

month = feb,

day = "20",

doi = "10.1093/nar/gky1187",

language = "English",

volume = "47",

pages = "1493--1504",

journal = "Nucleic Acids Research",

issn = "0305-1048",

publisher = "Oxford University Press",

number = "3",

}

TY - JOUR

T1 - The Leishmania PABP1-eIF4E4 interface

T2 - A novel 5′-3′ interaction architecture for trans-spliced mRNAs

AU - Dos Santos Rodrigues, Fabio Henrique

AU - Firczuk, Helena

AU - Breeze, Alexander L.

AU - Cameron, Alexander D.

AU - Walko, Martin

AU - Wilson, Andrew J.

AU - Zanchin, Nilson I.T.

AU - McCarthy, John E.G.

N1 - Funding Information: Author contributions: J.E.G.M., F.H.dS.R., H.F., A.L.B., A.C., N.I.T.Z. and A.J.W. designed the research; F.H.dS.R. and H.F. performed the research; M.W. contributed peptides; J.E.G.M., A.L.B. and A.C. analyzed the data; J.E.G.M. wrote the paper with contributions from F.H.dS.R, A.C., A.L.B. and A.J.W. In the School of Life Sciences at the University of Warwick we thank Deborah Brotherton for advice on protein production and purification, Avinash Punekar for crystallographic data collection at the Diamond Light Source (proposal mx14692), as well as Cleidi Zampronio and Alex Jones for help with mass spectrometry. Protein production and biomolecular interaction studies via surface plasmon resonance, microscale thermophoresis and fluorescence anisotropy were performed using equipment in the WISB Research Technology Facility. We acknowledge access to the Astbury Biostructure Laboratory BioNMR Facility (University of Leeds) and also thank Arnout Kalverda (BioNMR Facility) for NMR technical assistance. Medical Research Council funding [MR/N017447/1] to the Universities of Warwick and Leeds; Fundação Araucária funding to the Instituto Carlos Chagas (FIOCRUZParaná) [005/2016]; Brazilian Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the award of a Ciência sem Fronteiras (Science Without Borders) studentship to Fábio Henrique dos Santos Rodrigues. WISB is funded by the BBSRC and EPSRC [BB/M017982/1] and the University of Warwick. Funding to the Astbury Biostructure Laboratory BioNMR Facility from the Wellcome Trust [104920/Z/14/Z]; University of Leeds; EPSRC funding [EP/N035267/1 to M.W.]; peptide production, purification and characterization infrastructure supported by EPSRC [EP/N013573/1, EP/KO39292/1]; Wellcome Trust [WT097827/Z/11/A]. Funding for open access charge: University of Warwick. Funding Information: Medical Research Council funding [MR/N017447/1] to the Universities of Warwick and Leeds; Fundac¸ão Araucária funding to the Instituto Carlos Chagas (FIOCRUZ-Paraná) [005/2016]; Brazilian Conselho Nacional de De-senvolvimento Científico e Tecnológico (CNPq) for the award of a Ciência sem Fronteiras (Science Without Borders) studentship to Fábio Henrique dos Santos Ro-drigues. WISB is funded by the BBSRC and EPSRC [BB/M017982/1] and the University of Warwick. Funding to the Astbury Biostructure Laboratory BioNMR Facility from the Wellcome Trust [104920/Z/14/Z]; University of Leeds; EPSRC funding [EP/N035267/1 to M.W.]; peptide production, purification and characterization infrastructure supported by EPSRC [EP/N013573/1, EP/KO39292/1]; Wellcome Trust [WT097827/Z/11/A]. Funding for open access charge: University of Warwick. Conflict of interest statement. None declared. Publisher Copyright: © The Author(s) 2018.

PY - 2019/2/20

Y1 - 2019/2/20

N2 - 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 Xray 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.

AB - 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 Xray 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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DO - 10.1093/nar/gky1187

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JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 3

ER -

The Leishmania PABP1-eIF4E4 interface: A novel 5′-3′ interaction architecture for trans-spliced mRNAs

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