African trypanosomes are an excellent system for quantitative modelling of post-transcriptional mRNA control. Transcription is constitutive and polycistronic; individual mRNAs are excised by trans splicing and polyadenylation. We here measure mRNA decay kinetics in two life cycle stages, bloodstream and procyclic forms, by transcription inhibition and RNASeq. Messenger RNAs with short half-lives tend to show initial fast degradation, followed by a slower phase; they are often stabilized by depletion of the 5′–3′ exoribonuclease XRNA. Many longer-lived mRNAs show initial slow degradation followed by rapid destruction: we suggest that the slow phase reflects gradual deadenylation. Developmentally regulated mRNAs often show regulated decay, and switch their decay pattern. Rates of mRNA decay are good predictors of steady state levels for short mRNAs, but mRNAs longer than 3 kb show unexpectedly low abundances. Modelling shows that variations in splicing and polyadenylation rates can contribute to steady-state mRNA levels, but this is completely dependent on competition between processing and co-transcriptional mRNA precursor destruction.

Severe congenital neutropenia (SCN) is a BM failure syndrome with a high risk of progression to acute myeloid leukemia (AML). The underlying genetic changes involved in SCN evolution to AML are largely unknown. We obtained serial hematopoietic samples from an SCN patient who developed AML 17 years after the initiation of G-CSF treatment. Nextgeneration sequencing was performed to identify mutations during disease progression. In the AML phase, we found 12 acquired nonsynonymous mutations. Three of these, in CSF3R, LLGL2, and ZC3H18, co-occurred in a subpopulation of progenitor cells already in the early SCN phase. This population expanded over time, whereas clones harboring only CSF3R mutations disappeared from the BM. The other 9 mutations were only apparent in the AML cells and affected known AML-associated genes (RUNX1 and ASXL1) and chromatin remodelers (SUZ12 and EP300). In addition, a novel CSF3R mutation that conferred autonomous proliferation to myeloid progenitors was found. We conclude that progression from SCN to AML is a multistep process, with distinct mutations arising early during the SCN phase and others later in AML development. IntroductionSevere congenital neutropenia (SCN) is a BM failure syndrome characterized by strongly reduced neutrophil counts and recurrent, potentially life-threatening, opportunistic bacterial infections. Treatment with G-CSF elevates peripheral neutrophil counts and reduces the risk of infections. 1 Leukemic progression of SCN is a major concern, with an estimated overall cumulative incidence of approximately 20% after 15 years of G-CSF treatment. 2 Constitutional mutations in the gene encoding neutrophil elastase (ELANE) are common defects in SCN. 3 In addition, the acquisition of nonsense mutations in the gene encoding the G-CSF receptor (CSF3R) is a unique feature in SCN patients. [4][5][6][7] These mutations lead to the expression of truncated CSF3R proteins, also known as the ␦ forms. In cell-line models, truncated CSF3R proteins are hampered in transducing the signals required for proper neutrophil differentiation, confer increased proliferative responses to G-CSF treatment, but do not cause leukemia in mice. [4][5][6][8][9][10][11] CSF3R␦ mutations can be detected in approximately 30% of SCN patients. In some cases, distinct clones with different CSF3R␦ mutations are present for many years. 7,12 After evolution of SCN toward acute myeloid leukemia (AML), CSF3R␦ mutations are found in approximately 80% of patients. 12 Until now, all reported SCN/AML patients harboring a CSF3R␦ mutation in the SCN phase also carry this mutation in the leukemic phase. These observations suggest that leukemic progression in SCN follows a unique pattern, with CSF3R␦ mutations as an early event, followed by additional genetic and epigenetic events that are essential for full leukemic transformation. Chromosomal aberrations such as loss of chromosome 7 and gain of chromosome 21 are apparent in AML arising from SCN and other BM failure syndromes such as Fanconi anemia and Shwachman-Dia...

A mathematical model of glycolysis in bloodstream form Trypanosoma brucei was developed previously on the basis of all available enzyme kinetic data (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215). The model predicted correctly the fluxes and cellular metabolite concentrations as measured in non-growing trypanosomes and the major contribution to the flux control exerted by the plasma membrane glucose transporter. Surprisingly, a large overcapacity was predicted for hexokinase (HXK), phosphofructokinase (PFK), and pyruvate kinase (PYK). Here, we present our further analysis of the control of glycolytic flux in bloodstream form T. brucei. First, the model was optimized and extended with recent information about the kinetics of enzymes and their activities as measured in lysates of in vitro cultured growing trypanosomes. Second, the concentrations of five glycolytic enzymes (HXK, PFK, phosphoglycerate mutase, enolase, and PYK) in trypanosomes were changed by RNA interference. The effects of the knockdown of these enzymes on the growth, activities, and levels of various enzymes and glycolytic flux were studied and compared with model predictions. Data thus obtained support the conclusion from the in silico analysis that HXK, PFK, and PYK are in excess, albeit less than predicted. Interestingly, depletion of PFK and enolase had an effect on the activity (but not, or to a lesser extent, expression) of some other glycolytic enzymes. Enzymes located both in the glycosomes (the peroxisome-like organelles harboring the first seven enzymes of the glycolytic pathway of trypanosomes) and in the cytosol were affected. These data suggest the existence of novel regulatory mechanisms operating in trypanosome glycolysis.Trypanosomatid parasites (Trypanosoma and Leishmania) are responsible for serious diseases of mankind in tropical and subtropical countries worldwide. These diseases affect millions of people, and hundreds of millions are at risk of becoming infected. Unfortunately, available treatments are largely inadequate. Currently used drugs are inefficacious and toxic. There is a desperate need for new effective and safe drugs, particularly in view of the development and spreading of drug resistance (1).Glycolysis plays an important role in the energy metabolism of these protozoan organisms, notably of Trypanosoma brucei when it lives in the blood of its mammalian host, causing a disease called sleeping sickness or human African trypanosomiasis in man and nagana in cattle (2, 3). This bloodstream form of T. brucei is entirely dependent on the conversion of the blood sugar glucose into pyruvate for its ATP supply. Oxidative metabolism involving mitochondrial Krebs cycle enzymes and oxidative phosphorylation are largely repressed. Therefore, glycolysis has been perceived as a potentially good target for anti-trypanosome drugs. Moreover, the glycolytic pathway in trypanosomatids is organized in a unique manner: the majority of the glycolytic enzymes are sequester...

ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation. Fig. S1]. In glycolysis, ATP is first invested in 2 phosphorylation reactions, catalyzed by hexokinase (HXK) and phosphofructokinase (PFK). Only further downstream in the pathway the ATP is regained and then a surplus of ATP is generated. In glycerol metabolism, ATP is invested in the reaction catalized by glycerol kinase (GK) (Fig. 1).In the absence of specific regulation of HXK and PFK, the ATP produced by glycolysis could boost the flux through these enzymes above the capacity of the enzymes downstream, and hexose phosphates [glucose 6-phosphate (Glc6P), fructose 6-phosphate (Fru6P) and fructose 1,6-phosphate (Fru1,6BP)] would accumulate to extreme levels. By analogy to the turbo engine (which uses engine exhaust to boost performance), this property was called the ''turbo-design''of glycolysis (2). Many organisms avoid the negative side effects of the autocatalytic design of glycolysis by a tight feedback regulation of HXK and PFK, e.g., inhibition of HXK by Glc6P (3) or trehalose 6-phosphate (5). In yeast, deletion of trehalose-6-phosphate synthase leads to glucose toxicity, accumulation of hexose phosphates and rapid consumption of ATP (4). This ''turbo-explosion'' phenotype is rescued by reducing the expression of HXK (5) or glucose transporters (6).Trypanosoma brucei, the tropical parasite that causes the deadly African sleeping sickness, lacks feedback regulation of HXK and PFK (7-9). The parasite has a complex life cycle that alternates between insect and mammalian hosts; in the latter it lives in the bloodstream, supplied with a ready source of glucose. How then are trypanosomes protected against a possible turboexplosion of glycolysis?A key feature of trypanosome glycolysis is the compartmentation of the first 7 enzymes of glycolysis and 2 involved in glycerol metabolism inside peroxisome-like organelles called glycosomes (10). In the glycosome, ATP and redox levels are ba...