LeishIF3d is a non-canonical cap-binding protein in Leishmania

Abstract

Translation of most cellular mRNAs in eukaryotes proceeds through a cap-dependent pathway, whereby the cap-binding complex, eIF4F, anchors the pre-initiation complex at the 5′ end of mRNAs driving translation initiation. The genome of Leishmania encodes a large repertoire of cap-binding complexes that fulfill a variety of functions possibly involved in survival along the life cycle. However, most of these complexes function in the promastigote life form that resides in the sand fly vector and decrease their activity in amastigotes, the mammalian life form. Here we examined the possibility that LeishIF3d drives translation in Leishmania using alternative pathways. We describe a non-canonical cap-binding activity of LeishIF3d and examine its potential role in driving translation. LeishIF3d is required for translation, as reducing its expression by a hemizygous deletion reduces the translation activity of the LeishIF3d(+/−) mutant cells. Proteomic analysis of the mutant cells highlights the reduced expression of flagellar and cytoskeletal proteins, as reflected in the morphological changes observed in the mutant cells. Targeted mutations in two predicted alpha helices diminish the cap-binding activity of LeishIF3d. Overall, LeishIF3d could serve as a driving force for alternative translation pathways, although it does not seem to offer an alternative pathway for translation in amastigotes.

LeishIF4E2 is a cap‐binding protein that plays a role in Leishmania cell cycle progression

“…Although the mutant cells of the hemizygous mutant lines were smaller, round, and equipped with a shorter flagellum, they varied in their effect on global translation. For example, the hemizygous deletion of LeishIF4E2 did not affect the growth and global translation in the LeishIF4E2(+/−) mutant cells, unlike the hemizygous mutants of LeishIF4E3 27 and LeishIF3d 60 . Therefore, LeishIF4E2 could be involved in processes other than global translation, or it could possibly involve the translation of specific transcripts.…”

“…For example, the hemizygous deletion of LeishIF4E2 did not affect the growth and global translation in the LeishIF4E2(+/−) mutant cells, unlike the hemizygous mutants of LeishIF4E3 27 and LeishIF3d. 60 Therefore, LeishIF4E2 could be involved in processes other than global translation, or it could possibly involve the translation of specific transcripts. It is important to note that despite repeated attempts to delete the second allele of the LeishIF4E2 gene using the Blasticidin cassette on the G418 background of LeishIF4E2(+/−), a complete null mutant could not be generated, as the cells were unable to grow in the presence of Blasticidin and G418.…”

“…Hemizygous mutants were obtained for LeishIF4E2, 27 LeishIF4E3, 59 LeishIF4E6, 55 and LeishIF3d. 60 Although the mutant cells of the hemizygous mutant lines were smaller, round, and equipped with a shorter flagellum, they varied in their effect on global translation. For example, the hemizygous deletion of LeishIF4E2 did not affect the growth and global translation in the LeishIF4E2(+/−) mutant cells, unlike the hemizygous mutants of LeishIF4E3 27 and LeishIF3d.…”

“…However, in some cases, only a single allele could be removed, suggesting that the target gene could be essential. Hemizygous mutants were obtained for LeishIF4E2, 27 LeishIF4E3, 59 LeishIF4E6, 55 and LeishIF3d 60 . Although the mutant cells of the hemizygous mutant lines were smaller, round, and equipped with a shorter flagellum, they varied in their effect on global translation.…”