TheLeishmaniaPABP1–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 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.

Nutritional stress targets LeishIF4E-3 to storage granules that contain RNA and ribosome components in Leishmania

“…The N-terminal extension of LeishIF4E-4 was recently shown to be responsible for binding LeishPABP1 and a similar prediction was made for LeishIF4E-3 in that paper [50]. Our analysis highlights that LeishIF4E-3 and LeishPABP2 co-migrate to the same granules.…”

“…The latter has been implicated in elongation, rather than in initiation of translation [ 49 ]. The enriched fractions also contained LeishPABP2, but not LeishPABP1, which is part of the LeishIF4E-4 canonical complex [ 29 , 50 ]. The trypanosomatid ortholog TbPABP2 is also found in stress granules in T .…”

Regulation of gene expression in trypanosomatids: living with polycistronic transcription

“…RNAi results, combined with failed knock-out attempts, indicate that the EIF4E3- and EIF4E4-based complexes are essential for general translation and cell survival [174]. Interestingly, the N-terminal extension of Leishmania EIF4E4 associates directly with PABP1 [179], while the C-terminal conserved domain is responsible for recruiting EIF4G3. Whether EIF4G3 also interacts directly with PABP1 is controversial [168,179,180].…”

Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

“…Several structures of PAM2 motifs bound to MLLE domains have recently been determined [ 15 , 18 , 19 , 20 , 21 , 22 ]; see [ 17 ] for a review. Within those structures, the respective MLLE domain appears as a bundle of four or five α-helices.…”