The RNA-associated proteins MKT1 and MKT1L form alternative PBP1-containing complexes in Trypanosoma brucei

Abstract

Control of gene expression in kinetoplastids such as trypanosomes depends heavily on RNA-binding proteins that influence mRNA decay and translation. We previously showed that the trypanosome protein MKT1 forms a multicomponent protein complex: MKT1 interacts with PBP1, which in turn recruits LSM12 and poly(A)-binding protein. MKT1 is recruited to mRNAs by sequence-specific RNA-binding proteins, resulting in stabilization of the bound mRNA. We here show that PBP1, LSM12, and a 117-residue protein, XAC1 (Tb927.7.2780), are present in complexes that contain either MKT1 or an MKT1-like protein, MKT1L (Tb927.10.1490). All five proteins are present predominantly in the complexes, and we found evidence for a minor subset of complexes containing both MKT1 and MKT1L. XAC1-containing complexes reproducibly contained RNA-binding proteins that were previously found associated with MKT1. Moreover, XAC1- or MKT1- containing complexes specifically recruited one of the two poly(A)-binding proteins, PABP2, and one of the six cap-binding translation initiation complexes, EIF4E6–EIF4G5. Yeast two-hybrid assay results indicated that MKT1 directly interacts with EIF4G5. MKT1–PBP1 complexes can therefore interact with mRNAs via their poly(A) tails and caps, as well as through sequence-specific RNA-binding proteins. Correspondingly, MKT1 is associated with many mRNAs, although not with those encoding ribosomal proteins. Meanwhile, MKT1L resembles MKT1 at the C-terminus, but additionally features an N-terminal extension with low-complexity regions. Although MKT1L depletion inhibited cell proliferation, we found no evidence that it specifically interacts with RNA-binding proteins or mRNA. We speculate that MKT1L may compete with MKT1 for PBP1 binding and thereby modulate the function of MKT1-containing complexes.

The trypanosome Variant Surface Glycoprotein mRNA is stabilized by an essential unconventional RNA-binding protein

“…Evidence so far suggests that although both MKT1 and PBP1 have some intrinsic RNA-binding activity, they are recruited to specific mRNAs by various different RNA-binding proteins, resulting in enhanced mRNA abundance and translation (26,48,51). CFB2 showed clear-copurification with MKT1 and XAC1 (48,49). Correspondingly, both MKT1 and PBP1 were highly enriched in the VSG mRNP (Fig.…”

“…This may fine-tune CFB2 abundance to limit VSG synthesis to an ideal level, preventing secretory pathway overload. SKP1 was detectable in purified MKT1-containing complexes (48,49), so CFB2 may be able to interact with SKP1 and MKT1 simultaneously. We do not know whether CFB2 has additional ubiquitination targets.…”

“…The C-terminus of CFB2 includes a motif, RYRHDPY, which is required for the interaction of CFB2 with MKT1 (48,49) ( Supplementary Fig 3). T. brucei MKT1 forms a complex with PBP1, LSM12, XAC1 and PABP2 (48,49), and also preferentially recruits one of the six alternative cap-binding translation initiator factor complexes, EIF4E6-EIF4G5 (49,50) ( Fig 3D).…”

A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment

“…The essential gene encoding γ-glutamylcysteine synthetase (GSH1; Tb927.10.12370 ) ( 50 ), which is the rate-limiting step of trypanothione biosynthesis ( 26 , 38 , 42 , 51 ), was 191-fold overrepresented in melarsoprol survivors ( Table 2 ). Trypanothione is the primary intracellular target of melarsoprol, and overexpression of GSH1 in T. brucei and other trypanosomatids increases the concentration of intracellular trypanothione, resulting in melarsoprol resistance under laboratory conditions ( 52 ). In our hands, overexpression of GSH1 resulted in an approximately 1.5-fold increase in the relative EC 50 of melarsoprol ( Fig.…”

“…Hits categorized as gene expression represent a complex list including genes associated with splicing, posttranscriptional activation, and repression. XAC1 is an established posttranscriptional activator that does not bind mRNA directly but forms complexes with other poly(A)-binding proteins (e.g., MKT1 and PBP-1) (52,58). The gene encoding XAC1 was among the top hits, and its overexpression increased the EC 50 of melarsoprol (Fig.…”