Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei

Colasante, Claudia; Robles, Ana I.; Li, Chi-Ho; Schwede, Angela; Benz, Corinna; Voncken, Frank; Guilbride, D. Lys; Clayton, Christine

doi:10.1016/j.molbiopara.2006.11.003

Cited by 50 publications

(44 citation statements)

References 39 publications

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“…Amounts and Turnover of PGK and TUBA mRNAs-Northern blotting with PGK standards (Fig. 1A) showed that bloodstream form trypanosomes contained 12.1 Ϯ 7.7 (S.D., n ϭ 5) molecules of PGKC mRNA (this study) and 15 times less PGKB mRNA per cell (33). In procyclic trypanosomes, there were 18 Ϯ 2 (S.D., n ϭ 5) PGKB mRNAs per cell, and PGKC mRNA was not detectable, as expected from previous reports (Gibson et al (27); Colasante et al (33)).…”

Section: Resultssupporting

confidence: 58%

“…However, correct localization of PGK activity, and hence regulation of PGKB and PGKC expression, are vital, because cytosolic PGK activity causes growth arrest of bloodstream form trypanosomes (31). Qualitatively, the expression patterns of the isoenzymes can be explained by the different stabilities of their mature mRNAs, depending on their 3Ј-untranslated regions (UTRs) (32)(33)(34)(35)(36). There might be additional regulation at the levels of splicing (37,38), translation (39), and variations in protein stability, but the quantitative importance of these processes is unknown, and there has not been a methodology even to establish this.…”

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Control and Regulation of Gene Expression

Haanstra

Stewart

Luu

et al. 2008

Journal of Biological Chemistry

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Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to make sufficient amounts of the isoenzyme with the correct localization, which processes (transcription, splicing, and RNA degradation) control the levels of mRNAs, and how the organism regulates the switch in isoform expression. For this, we combined new quantitative measurements of phosphoglycerate kinase mRNA abundance, RNA precursor stability, trans splicing, and ribosome loading with published data and made a kinetic computer model. For the analysis of regulation we extended regulation analysis. Although phosphoglycerate kinase mRNAs are present at surprisingly low concentrations (e.g. 12 molecules per cell), its protein is highly abundant. Substantial control of mRNA and protein levels was exerted by both mRNA synthesis and degradation, whereas splicing and precursor degradation had little control on mRNA and protein concentrations. Yet regulation of mRNA levels does not occur by transcription, but by adjusting mRNA degradation. The contribution of splicing to regulation is negligible, as for all cases where splicing is faster than RNA precursor degradation.The flux through a metabolic pathway depends on the kinetic characteristics and concentrations of the constituent enzymes, on the levels of co-enzymes, and on their compartmentation. The concentrations of the enzymes in turn depend on the rates of transcription, processing, nuclear export, translation, degradation of the mRNA, and protein processing and degradation. Because each of these levels can in principle be regulated, the challenge is how to analyze the behavior of such a complex system in terms of the underlying processes.Metabolic control analysis (MCA) 4 and extensions that include gene expression is a powerful approach for the analysis of complex biochemical networks (1-4). In MCA, the control exerted by an enzyme on a concentration of any substance X (5) is quantified by its concentration control coefficient, which is defined as the percentage increase of the steady-state concentration of X that results from a 1% activation of the enzyme of interest. The sum of the concentration control coefficients of all the enzymes in the network is 0. This reflects that (i) activation of some enzymes increases the concentration of X, whereas activation of other enzymes should reduce the concentration of X (these enzymes have negative concentration control coefficients), and (ii) the positive controls together are equal to the negative controls. The principles of MCA do not only apply to metabolic pathways; they can also be used and extended to dissect the control distribution in regulatory pathways beyond steady state (for example see Refs.6 and 7) or gene expression cascades (for example see e.g. Ref. 8). Earlier MCA, as also applied to trypanosomes, has looked more at the control of fluxes, showing that usually flux control coefficients are smaller than 1, because several enzymes partially...

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Section: Resultssupporting

confidence: 58%

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confidence: 99%

Control and Regulation of Gene Expression

Haanstra

Stewart

Luu

et al. 2008

Journal of Biological Chemistry

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“…Unmethylated SLRNA is not a substrate for trans splicing (Ullu and Tschudi 1991). Five minutes later, when we expected reduced cap methylation, we added Actinomycin D (Colasante et al 2007).…”

Section: Resultsmentioning

confidence: 99%

The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation

Fadda

Färber

Droll

et al. 2013

RNA

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The degradation of eukaryotic mRNAs can be initiated by deadenylation, decapping, or endonuclease cleavage. This is followed by 5 ′ -3 ′ degradation by homologs of Xrn1, and/or 3 ′ -5 ′ degradation by the exosome. We previously reported that, in African trypanosome Trypanosoma brucei, most mRNAs are deadenylated prior to degradation, and that depletion of the major 5 ′ -3 ′ exoribonuclease XRNA preferentially stabilizes unstable mRNAs. We now show that depletion of either CAF1 or CNOT10, two components of the principal deadenylation complex, strongly inhibits degradation of most mRNAs. RNAi targeting another deadenylase, PAN2, or RRP45, a core component of the exosome, preferentially stabilized mRNAs with intermediate half-lives. RRP45 depletion resulted in a 5 ′ bias of mRNA sequences, suggesting action by a distributive 3 ′ -5 ′ exoribonuclease. Results suggested that the exosome is involved in the processing of trypanosome snoRNAs. There was no correlation between effects on half-lives and on mRNA abundance.

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“…To measure the half-life of the mRNA during silencing, uninduced cells and cells 3 d after induction were concentrated. After a few minutes of recovery, the cells were treated with sinefungin, which was shown to inhibit trans-splicing and to assist in shutting-off mRNA production; hence, this agent improves the quality of experiments that determine the half-life of mRNAs (Colasante et al 2007). Sinefungin was added to the cells for 10 min, then actinomycin D was added, and RNA was extracted from the cells at the indicated time points.…”

Section: Validation Of the Microarray Datamentioning

confidence: 99%

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

Stern¹,

Gupta²,

Salmon‐Divon³

et al. 2009

RNA

107

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Trypanosomatid genomes encode for numerous proteins containing an RNA recognition motif (RRM), but the function of most of these proteins in mRNA metabolism is currently unknown. Here, we report the function of two such proteins that we have named PTB1 and PTB2, which resemble the mammalian polypyrimidine tract binding proteins (PTB). RNAi silencing of these factors indicates that both are essential for life. PTB1 and PTB2 reside mostly in the nucleus, but are found in the cytoplasm, as well. Microarray analysis performed on PTB1 and PTB2 RNAi silenced cells indicates that each of these factors differentially affects the transcriptome, thus regulating a different subset of mRNAs. PTB1 and PTB2 substrates were categorized bioinformatically, based on the presence of PTB binding sites in their 59 and 39 flanking sequences. Both proteins were shown to regulate mRNA stability. Interestingly, PTB proteins are essential for trans-splicing of genes containing C-rich polypyrimidine tracts. PTB1, but not PTB2, also affects cis-splicing. The specificity of binding of PTB1 was established in vivo and in vitro using a model substrate. This study demonstrates for the first time that trans-splicing of only certain substrates requires specific factors such as PTB proteins for their splicing. The trypanosome PTB proteins, like their mammalian homologs, represent multivalent RNA binding proteins that regulate mRNAs from their synthesis to degradation.

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Regulated expression of glycosomal phosphoglycerate kinase in Trypanosoma brucei

Cited by 50 publications

References 39 publications

Control and Regulation of Gene Expression

Control and Regulation of Gene Expression

The roles of 3′-exoribonucleases and the exosome in trypanosome mRNA degradation

Multiple roles for polypyrimidine tract binding (PTB) proteins in trypanosome RNA metabolism

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