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.
Highlights
Transcription initiation is a crucial point of gene expression regulation in most eukaryotes
ZC3H11 binds to the 39-UTRs of mRNAs that are required for survival after heat shock [15], whereas ZC3H20 binds to a subset of mRNAs that is preferentially expressed in procyclic forms [16, 17]
Stabilization of mRNAs by ZC3H11 and ZC3H20 requires binding to MKT1, which in turn recruits PBP1, LSM12, and poly(A)-binding proteins (PABPs), especially PABP2 [16, 18]
Summary
T. brucei XAC1 is 117 residues long and has no annotated functional domains (Fig. 1A). MKT1L was robustly associated with XAC1, LSM12, and PBP1, with some MKT1 detected (Fig. 2D) (Table S2) These results confirmed that the complexes are present in procyclic forms. 0.05; No, not detected; Yes*, ZC3H20 has RNA-binding domains and binds to mRNAs in procyclic forms [17] but has low abundance in bloodstream forms so was not in the mRNP proteome); 5, effect of tethering to a reporter (A, activates as a full-length protein [39]; (A), activated in a high-throughput screen of protein fragments [38]; *, shown or confirmed in this work; (a), has been shown by other means to stabilize its target mRNAs [45]; R, represses as full-length protein; 0, no effect detected; 6, percentage coverage in a single purification of MKT1-TAP [18]; 7, interaction with MKT1 in yeast-two-hybrid assay (Y2H) (individual or screen) (number of different interacting fragments detected [18]); 8, log p value for co-purification with XAC1-TAP; 9, enrichment with XAC1-TAP. Values for columns 8 and 9 were obtained using PERSEUS
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