TY - JOUR
T1 - Distinct mRNA and protein interactomes highlight functional differentiation of major eIF4F-like complexes from Trypanosoma brucei
AU - Bezerra, Maria J. R.
AU - Moura, Danielle M. N.
AU - Freire, Eden R.
AU - Holetz, Fabiola B.
AU - Reis, Christian R. S.
AU - Monteiro, Tallyta T. S.
AU - Pinto, Adriana R. S.
AU - Zhang, Ning
AU - Rezende, Antonio M.
AU - Pereira-Neves, Antonio
AU - Figueiredo, Regina C. B. Q.
AU - Clayton, Christine
AU - Field, Mark C.
AU - Carrington, Mark
AU - de Melo Neto, Osvaldo P.
N1 - Funding Information:
In Brazil, this work was mainly supported by grants provided by the Brazilian Funding agencies FACEPE (APQ1662-2.02/15) and CNPq (401888/2013-4; 400789/2019-1) awarded to OdM. This study was also partially financed in part by the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior—Brazil (CAPES)—Finance Code 001. The SOLID sequencing work was supported by a grant from FACEPE to FH (DCR 0032-2.13/09; APQ-0090-2.13/09). More recent work was funded by a German Deutsche Forschungsgemeinschaft grant (CL 112/30-1) awarded to CC and OdM.OdMisalsorecipient of aCNPqfellowship (310032/ 2019-9). TM was a recipient of an undergraduate PIBIC/ Fiocruz/CNPq scholarship while MB is a recipient of a CAPES/FACEPE/PNPD grant (APQ-0876-2.02/16) and EF was a recipient of a BJT fellowship awarded by CNPq (401282/2014-7). MC and MF are Wellcome Investigators (grants 217138/Z/19/Z and 204697/Z/16/Z, respectively).
Copyright:
© 2022 Bezerra, Moura, Freire, Holetz, Reis, Monteiro, Pinto, Zhang, Rezende, Pereira-Neves, Figueiredo, Clayton, Field, Carrington and de Melo Neto.
PY - 2022/10/7
Y1 - 2022/10/7
N2 - Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.
AB - Gene expression in pathogenic protozoans of the family Trypanosomatidae has several novel features, including multiple eIF4F-like complexes involved in protein synthesis. The eukaryotic eIF4F complex, formed mainly by eIF4E and eIF4G subunits, is responsible for the canonical selection of mRNAs required for the initiation of mRNA translation. The best-known complexes implicated in translation in trypanosomatids are based on two related pairs of eIF4E and eIF4G subunits (EIF4E3/EIF4G4 and EIF4E4/EIF4G3), whose functional distinctions remain to be fully described. Here, to define interactomes associated with both complexes in Trypanosoma brucei procyclic forms, we performed parallel immunoprecipitation experiments followed by identification of proteins co-precipitated with the four tagged eIF4E and eIF4G subunits. A number of different protein partners, including RNA binding proteins and helicases, specifically co-precipitate with each complex. Highlights with the EIF4E4/EIF4G3 pair include RBP23, PABP1, EIF4AI and the CRK1 kinase. Co-precipitated partners with the EIF4E3/EIF4G4 pair are more diverse and include DRBD2, PABP2 and different zinc-finger proteins and RNA helicases. EIF4E3/EIF4G4 are essential for viability and to better define their role, we further investigated their phenotypes after knockdown. Depletion of either EIF4E3/EIF4G4 mRNAs lead to aberrant morphology with a more direct impact on events associated with cytokinesis. We also sought to identify those mRNAs differentially associated with each complex through CLIP-seq with the two eIF4E subunits. Predominant among EIF4E4-bound transcripts are those encoding ribosomal proteins, absent from those found with EIF4E3, which are generally more diverse. RNAi mediated depletion of EIF4E4, which does not affect proliferation, does not lead to changes in mRNAs or proteins associated with EIF4E3, confirming a lack of redundancy and distinct roles for the two complexes.
KW - PABP
KW - RNA processing
KW - eIF4E
KW - eIF4G
KW - protein synthesis
KW - translation initiation
KW - trypanosome
UR - http://www.scopus.com/inward/record.url?scp=85140289806&partnerID=8YFLogxK
U2 - 10.3389/fmolb.2022.971811
DO - 10.3389/fmolb.2022.971811
M3 - Article
C2 - 36275617
SN - 2296-889X
VL - 9
JO - Frontiers in Molecular Biosciences
JF - Frontiers in Molecular Biosciences
M1 - 971811
ER -