Clinically relevant benzoxaboroles inhibit mRNA processing in Trypanosoma brucei.

Objective: The cleavage and polyadenylation endonuclease CPSF73 is thought to be the target of the anti-trypanosomal benzoxaboroles AN7973, acoziborole and AN11736. We previously showed that AN7973 inhibits mRNA processing. We here investigated whether the drug candidates acoziborole (for human sleeping sickness) and AN11736 (for nagana in cattle) have the same effect.

Sequences and proteins that influence mRNA processing in Trypanosoma brucei: Evolutionary conservation of SR-domain and PTB protein functions.

Background: Spliced leader trans splicing is the addition of a short, capped sequence to the 5' end of mRNAs. It is widespread in eukaryotic evolution, but factors that influence trans splicing acceptor site choice have been little investigated. In Kinetoplastids, all protein-coding mRNAs are 5' trans spliced.

Roles and interactions of the specialized initiation factors EIF4E2, EIF4E5, and EIF4E6 in Trypanosoma brucei: EIF4E2 maintains the abundances of S-phase mRNAs.

Trypanosoma brucei has six versions of the cap-binding translation initiation factor EIF4E. We investigated the functions of EIF4E2, EIF4E3, EIF4E5, and EIF4E6 in bloodstream forms. We confirmed the protein associations previously found in procyclic forms and detected specific copurification of some RNA-binding proteins.

Gel shift experiments with fragments of the Trypanosoma brucei RNA-binding protein RBP10.

Objective: Trypanosoma brucei is a parasite of mammals and Tsetse flies, and control of mRNA stability is critical for parasite survival in the two different hosts. T. brucei RBP10 is a protein with a single RNA Recognition Motif (RRM) which is expressed only in the mammalian (bloodstream) form.

The RNA-binding protein DRBD18 ensures correct mRNA splicing and polyadenylation patterns.

The parasite grows as bloodstream forms in mammals, and as procyclic forms in tsetse flies. Transcription is polycistronic, all mRNAs are spliced, and polyadenylation sites are defined by downstream splicing signals. Expression regulation therefore depends heavily on post-transcriptional mechanisms.

Several different sequences are implicated in bloodstream-form-specific gene expression in Trypanosoma brucei.

The parasite Trypanosoma brucei grows as bloodstream forms in mammalian hosts, and as procyclic forms in tsetse flies. In trypanosomes, gene expression regulation depends heavily on post-transcriptional mechanisms. Both the RNA-binding protein RBP10 and glycosomal phosphoglycerate kinase PGKC are expressed only in mammalian-infective forms.

Interactions of the zinc-finger-domain protein ZC3H28.

In Trypanosoma brucei and related Kinetoplastids, regulation of gene expression occurs mostly post-transcriptionally, and RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Trypanosoma brucei ZC3H28 is a 114 KDa cytoplasmic mRNA-binding protein with a single C(x)7C(x)5C(x)sH zinc finger at the C-terminus and numerous proline-, histidine- or glutamine-rich regions. ZC3H28 is essential for normal bloodstream-form trypanosome growth, and when tethered to a reporter mRNA, ZC3H28 increased reporter mRNA and protein levels.

The EIF4E1-4EIP cap-binding complex of Trypanosoma brucei interacts with the terminal uridylyl transferase TUT3.

Most transcription in Trypanosoma brucei is constitutive and polycistronic. Consequently, the parasite relies on post-transcriptional mechanisms, especially affecting translation initiation and mRNA decay, to control gene expression both at steady-state and for adaptation to different environments. The parasite has six isoforms of the cap-binding protein EIF4E as well as five EIF4Gs.

In vitro culture of freshly isolated Trypanosoma brucei brucei bloodstream forms results in gene copy-number changes.

Most researchers who study unicellular eukaryotes work with an extremely limited number of laboratory-adapted isolates that were obtained from the field decades ago, but the effects of passage in laboratory rodents, and adaptation to in vitro culture, have been little studied. For example, the vast majority of studies of Trypanosoma brucei biology have concentrated on just two strains, Lister 427 and EATRO1125, which were taken from the field over half a century ago and have since have undergone innumerable passages in rodents and culture. We here describe two new Trypanosoma brucei brucei strains.

4E Interacting Protein as a Potential Novel Drug Target for Nucleoside Analogues in .

Human African trypanosomiasis is a neglected parasitic disease for which the current treatment options are quite limited. Trypanosomes are not able to synthesize purines de novo and thus solely depend on purine salvage from the host environment. This characteristic makes players of the purine salvage pathway putative drug targets.

Functional insights from a surface antigen mRNA-bound proteome.

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.

Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase.

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line.

Insights into the functions and RNA binding of ZC3H22, RBP9 and DRBD7.

Trypanosoma brucei is unusually reliant on mRNA-binding proteins to control mRNA fate, because its protein-coding genes lack individual promoters. We here focus on three trypanosome RNA-binding proteins. ZC3H22 is specific to Tsetse fly forms, RBP9 is preferentially expressed in bloodstream forms; and DRBD7 is constitutively expressed.

A potential role for a novel ZC3H5 complex in regulating mRNA translation in .

In and related kinetoplastids, gene expression regulation occurs mostly posttranscriptionally. Consequently, RNA-binding proteins play a critical role in the regulation of mRNA and protein abundance. Yet, the roles of many RNA-binding proteins are not understood.

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

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.

Polysome Profiling and Metabolic Labeling Methods to Measure Translation in Trypanosoma brucei.

The amount of a protein that is made in a cell is determined not only by the corresponding mRNA level but also by the efficiency with which the mRNA is translated. Very powerful transcriptome-wide methods are available to analyze both the density of ribosomes on each mRNA and the rate at which polypeptides are elongated. However, for many research questions, simpler, less expensive methods are more suitable.

High-Throughput Sequencing for Trypanosome Transcriptome Characterization.

High-throughput sequencing of cDNA (RNASeq) is now the method of choice for analysis of transcriptomes. This chapter details important considerations in the design of RNASeq experiments for kinetoplastids grown in culture or experimental animals. It contains protocols for obtaining parasites from rodents, and for removal of rRNA from total RNA.

The endoplasmic reticulum-associated mRNA-binding proteins ERBP1 and ERBP2 interact in bloodstream-form .

Kinetoplastids rely heavily on post-transcriptional mechanisms for control of gene expression, and on RNA-binding proteins that regulate mRNA splicing, translation and decay. ERBP1 (Tb927.10.

Blood signatures for second stage human African trypanosomiasis: a transcriptomic approach.

Background: Rhodesiense sleeping sickness is caused by infection with T. b rhodesiense parasites resulting in an acute disease that is fatal if not treated in time. The aim of this study was to understand the global impact of active T.

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect-form Trypanosoma brucei.

ZC3H20 and ZC3H21 are related trypanosome proteins with two C(x) C(x) C(x) H zinc finger motifs. ZC3H20 is present at a low level in replicating mammalian-infective bloodstream forms, but becomes more abundant when they undergo growth arrest at high density; ZC3H21 appears only in the procyclic form of the parasite, which infects Tsetse flies. Each protein binds to several hundred mRNAs, with overlapping but not identical specificities.

Regulation of gene expression in trypanosomatids: living with polycistronic transcription.

In trypanosomes, RNA polymerase II transcription is polycistronic and individual mRNAs are excised by trans-splicing and polyadenylation. The lack of individual gene transcription control is compensated by control of mRNA processing, translation and degradation. Although the basic mechanisms of mRNA decay and translation are evolutionarily conserved, there are also unique aspects, such as the existence of six cap-binding translation initiation factor homologues, a novel decapping enzyme and an mRNA stabilizing complex that is recruited by RNA-binding proteins.

Novel Observations Concerning Differentiation of Bloodstream-Form Trypanosomes to the Form That Is Adapted for Growth in Tsetse Flies.

Salivarian trypanosomes grow in mammals, where they depend on glucose, and as procyclic forms in tsetse flies, where they metabolize proline. Differentiation of bloodstream forms to nongrowing stumpy forms, and to procyclic forms, has been studied extensively, but reconciling the results is tricky because investigators have used parasites with various differentiation competences and different media for procyclic-form culture. Standard protocols include lowering the temperature to 27°C, adding a tricarboxylic acid, and transferring the parasites to high-proline medium, often including glucose.

Stress susceptibility in Trypanosoma brucei lacking the RNA-binding protein ZC3H30.

Trypanosomes rely on post-transcriptional mechanisms and mRNA-binding proteins for control of gene expression. Trypanosoma brucei ZC3H30 is an mRNA-binding protein that is expressed in both the bloodstream form (which grows in mammals) and the procyclic form (which grows in the tsetse fly midgut). Attachment of ZC3H30 to an mRNA causes degradation of that mRNA.

The trypanocidal benzoxaborole AN7973 inhibits trypanosome mRNA processing.

Kinetoplastid parasites-trypanosomes and leishmanias-infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis.