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

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 suppressive cap-binding complex factor 4EIP is required for normal differentiation.

Trypanosoma brucei live in mammals as bloodstream forms and in the Tsetse midgut as procyclic forms. Differentiation from one form to the other proceeds via a growth-arrested stumpy form with low messenger RNA (mRNA) content and translation. The parasites have six eIF4Es and five eIF4Gs. EIF4E1 pairs with the mRNA-binding protein 4EIP but not with any EIF4G. EIF4E1 and 4EIP each inhibit expression when tethered to a reporter mRNA, but while tethered EIF4E1 suppresses only when 4EIP is present, suppression by tethered 4EIP does not require the interaction with EIF4E1. In growing bloodstream forms, 4EIP is preferentially associated with unstable mRNAs. Bloodstream- or procyclic-form trypanosomes lacking 4EIP have only a marginal growth disadvantage. Bloodstream forms without 4EIP are, however, defective in translation suppression during stumpy-form differentiation and cannot subsequently convert to growing procyclic forms. Intriguingly, the differentiation defect can be complemented by a truncated 4EIP that does not interact with EIF4E1. In contrast, bloodstream forms lacking EIF4E1 have a growth defect, stumpy formation seems normal, but they appear unable to grow as procyclic forms. We suggest that 4EIP and EIF4E1 fine-tune mRNA levels in growing cells, and that 4EIP contributes to translation suppression during differentiation to the stumpy form.

Functional insights from a surface antigen mRNA-bound proteome.

Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites' variant surface glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2 (cyclin F-box protein 2), an unconventional RNA-binding protein with an F-box domain, was specifically enriched with VSG mRNA. We demonstrate that CFB2 is essential for VSG mRNA stability, describe cis acting elements within the VSG 3'-untranslated region that regulate the interaction, identify trans-acting factors that are present in the VSG messenger ribonucleoprotein particle, and mechanistically explain how CFB2 stabilizes the mRNA of this key pathogenicity factor. Beyond T. brucei, the mRNP purification approach has the potential to supply detailed biological insight into metabolism of relatively abundant mRNAs in any eukaryote.

Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.

Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.

Conversion of procyclic-form Trypanosoma brucei to the bloodstream form by transient expression of RBP10.

Bloodstream-form Trypanosoma brucei can lose the ability to differentiate to the procyclic form during prolonged in vitro culture. This can pose a problem during complicated genetic manipulation experiments, especially when the differentiation phenotype is under investigation. Ideally, to preserve differentiation competence, parasites should be cycled after every genetic manipulation step. Conversion of bloodstream-form Trypanosoma brucei to the procyclic form in vitro is routine, but conversion of procyclic forms to bloodstream forms has hitherto only been achieved in transgenic parasites with tetracycline-inducible expression of proteins with RNA-binding domains - either RBP6 or RBP10. This method, however, requires use of a selectable marker which might be needed for other purposes, and restricts options for tetracycline-inducible expression or repression of other genes. A simple method for inter-conversion that does not require permanent genetic manipulation would therefore be useful. Induced expression of RBP10 in procyclic forms gives faster differentiation than expression of RBP6, with a switch towards bloodstream forms within 48h. We here show that bloodstream forms can be obtained by transient transfection of procyclic forms with a circular plasmid designed for expression of RBP10 from an rRNA promoter. This method enables routine cycling of T. brucei without permanent genetic manipulation.