Proteomics Uncovers Novel Components of an Interactive Protein Network Supporting RNA Export in Trypanosomes

Alexandre Haruo Inoue,Erin R. Butterfield,Patricia Ferreira Domingues,Mark C. Field,Cordula Boehm,Ricardo Canavate del Pino,Newton Medeiros Vidal,Andréa Rodrigues Ávila,Priscila Mazzocchi Hiraiwa,Mariana Serpeloni,Adriana Ludwig

doi:10.1016/j.mcpro.2022.100208

Abstract

In trypanosomatids, transcription is polycistronic and all mRNAs are processed by trans-splicing, with export mediated by noncanonical mechanisms. Although mRNA export is central to gene regulation and expression, few orthologs of proteins involved in mRNA export in higher eukaryotes are detectable in trypanosome genomes, necessitating direct identification of protein components. We previously described conserved mRNA export pathway components in Trypanosoma cruzi, including orthologs of Sub2, a component of the TREX complex, and eIF4AIII (previously Hel45), a core component of the exon junction complex (EJC). Here, we searched for protein interactors of both proteins using cryomilling and mass spectrometry. Significant overlap between TcSub2 and TceIF4AIII-interacting protein cohorts suggests that both proteins associate with similar machinery. We identified several interactions with conserved core components of the EJC and multiple additional complexes, together with proteins specific to trypanosomatids. Additional immunoisolations of kinetoplastid-specific proteins both validated and extended the superinteractome, which is capable of supporting RNA processing from splicing through to nuclear export and cytoplasmic events. We also suggest that only proteomics is powerful enough to uncover the high connectivity between multiple aspects of mRNA metabolism and to uncover kinetoplastid-specific components that create a unique amalgam to support trypanosome mRNA maturation.

Highlights

Trypanosoma cruzi is the causative agent of Chagas disease, a significant infection of humans and animals in Latin America where ten million people are estimated to be infected [1]
Following analysis by SDS-PAGE (Fig. 1A), where we confirmed the enrichment of the tagged-proteins, TcSub2 and TceIF4AIII isolates were analyzed by mass spectrometry and protein identifications filtered according to exponentially-modified protein abundance index
exon-junction complex (EJC) proteins interact with the T. cruzi mRNA export pathway We previously described the T. cruzi ortholog of eIF4AIII [60], and phylogenetic tree indicated that TceIF4AIII grouped into the eIF4AIII clade. eIF4AIII is a core component of the metazoan EJC and functions in multiple pathways of mRNA metabolism [41], while the S. cerevisiae ortholog, Fal1, is involved in 40Sribosomal-subunit biogenesis [103]

Summary

Introduction

Trypanosoma cruzi is the causative agent of Chagas disease, a significant infection of humans and animals in Latin America where ten million people are estimated to be infected [1]. RNA granules at the nuclear periphery may regulate mRNA fate by preventing mis-processed mRNAs from reaching the translation machinery [26, 27] It remains unclear how these complexes interact with the nuclear components involved in RNA export. Trypanosomes can initiate mRNA nuclear export co-transcriptionally and initiation of export does not depend on completion of trans-splicing, suggesting that trypanosomes regulate completion as opposed to initiation of export This is supported by the divergence of the cytoplasmic face composition of the NPC in trypanosomes [56] together with cytoplasmic nuclear peripheral granules (NPGs) but the components of both the NPC and export machinery responsible await identification [27]. We identified a kinetoplastid-specific NTF2-like protein that interacts with Mex, crucial for mRNA export These observations indicate considerable divergence in the composition and function of trypanosome mRNA export factors

Experimental Procedures

Results

Discussion