Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

Jha, Bhaskar Anand; Fadda, Abeer A.; Merce, Clementine; Mugo, Elisha; Droll, Dorothea; Clayton, Christine

doi:10.1128/ec.00018-14

Cited by 24 publications

(31 citation statements)

References 73 publications

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“…For this, we performed in vivo cross‐linking followed by poly(A) RNA enrichment (Castello et al ., ). To test whether the eluted proteins were enriched for bona fide RBPs, we first carried out western blotting to detect the well‐known RBPs: PABP1 (Singh et al ., ), PUF2 (Jha et al ., ), DRBD3 (Estevez, ) and the potential binder RBP10 (Wurst et al ., ). As expected, all proteins were enriched in cross‐linked (CL) samples and undetectable in controls (nonirradiated cells) (Fig.…”

Section: Resultsmentioning

confidence: 99%

Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA‐binding proteome

Lueong

Merce

Fischer

et al. 2016

Molecular Microbiology

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113

206

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Control of gene expression at the post-transcriptional level is essential in all organisms, and RNA-binding proteins play critical roles from mRNA synthesis to decay. To fully understand this process, it is necessary to identify the complete set of RNA-binding proteins and the functional consequences of the protein-mRNA interactions. Here, we provide an overview of the proteins that bind to mRNAs and their functions in the pathogenic bloodstream form of Trypanosoma brucei. We describe the production of a small collection of open-reading frames encoding proteins potentially involved in mRNA metabolism. With this ORFeome collection, we used tethering to screen for proteins that play a role in post-transcriptional control. A yeast two-hybrid screen showed that several of the discovered repressors interact with components of the CAF1/NOT1 deadenylation complex. To identify the RNA-binding proteins, we obtained the mRNA-bound proteome. We identified 155 high-confidence candidates, including many not previously annotated as RNA-binding proteins. Twenty seven of these proteins affected reporter expression in the tethering screen. Our study provides novel insights into the potential trypanosome mRNPs composition, architecture and function.

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Section: Resultsmentioning

confidence: 99%

Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA‐binding proteome

Lueong

Merce

Fischer

et al. 2016

Molecular Microbiology

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113

206

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“…Tethering of PUF4 fragments yielded no evidence that it has suppressive function [4], but several other PUFs functioned only at full length [3]. The only other cytoplasmic PUF protein that is known to have suppressive function is PUF2, which is essential in bloodstream forms [10]; its binding specificity is unknown. PUF2 probably has an abundance similar to that of PUF3 [8] so a period of adaptation might be required if it were to compensate for the absence of PUF3.…”

Section: Discussionmentioning

confidence: 99%

“…Only PUF2, 3, 4, and 6 have the canonical 8 pumilio repeats, and all but PUF3 (Tb927. 10.310) have low complexity or coiled coil motifs. Most of the PUF proteins are cytosolic; the exceptions are PUF7, PUF8 and PUF10, which are found in the nucleolus [2].…”

Section: Introductionmentioning

confidence: 99%

Expression of normal levels of the Pumilio domain protein PUF3 is required for optimal growth of Trypanosoma brucei

Marucha

Clayton

2019

Preprint

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The Trypanosoma brucei pumilio domain protein PUF3 is a cytosolic mRNA-binding protein that suppresses expression when tethered to a reporter mRNA. PUF3 was not essential in bloodstream-form or procyclic-form trypanosomes, and RNAi in bloodstream forms had no significant effect on the transcriptome. There was little evidence for specific binding of PUF3 to bloodstream-form mRNAs and mass spectrometry revealed no binding partners that might explain its suppressive activity. Ectopic expression of Cterminally tagged PUF3 in procyclic forms impaired viability. Reduction of PUF3 in bloodstream forms caused a slight growth defect and slightly delayed differentiation to the procyclic form, but the cells lost both defects upon prolonged cultivation. Procyclic forms without PUF3 grew somewhat slower than wild-type, but were able to transform to bloodstream forms after induced expression of the bloodstream-form RNA-binding protein RBP10. We suggest that PUF3 is implicated in fine-tuning gene expression. Since PUF3 is present throughout the Kinetoplastida, selection for its retention may occur within the arthropod vector.

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“…Such a hypothesis is attractive because VSG represents the most abundant mRNA and protein in the cell with both present at approximately 10% of total cell-load [3]. Shorter genes have also been reported to produce more abundant mRNAs in T. brucei [25,26], potentially facilitating expression of shorter genes. It is also important to point out that even small differences in the growth rate will cause measurable differences at the population level, due to exponential growth and progression to densities reaching >10 7 /ml of blood in mice [8,27].…”

Section: Discussionmentioning

confidence: 99%

Faster growth with shorter antigens explains a VSG hierarchy during African trypanosome infections: a feint attack by parasites

Liu

Albergante

Horn

et al. 2017

Preprint

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The parasitic African trypanosome, Trypanosoma brucei, evades the adaptive host immune response by a process of antigenic variation that involves the clonal switching of variant surface glycoproteins (VSGs). The VSGs that periodically come to dominate in vivo display a hierarchy, but how this hierarchy arises is not well-understood. Combining publicly available genetic data with mathematical modelling, we report a VSG-length-dependent hierarchical timing of clonal VSG dominance in a mouse model, revealing an inverse correlation between VSG length and trypanosome growth-rate. Our analysis indicates that, among parasites switching to new VSGs, those expressing shorter VSGs preferentially accumulate to a detectable level that is sufficient to trigger an effective immune response. Subsequent elimination of faster-growing parasites then allows slower parasites with longer VSGs to accumulate. This interaction between the host and parasite is able by itself to explain the temporal distribution of VSGs observed in vivo. Thus, our findings reveal a length-dependent hierarchy that operates during T. brucei infection, representing a 'feint attack' diversion tactic utilised during infection by these persistent parasites to out-maneuver the host immune system. Significance Statement (120 words)The protozoan parasite Trypanosoma brucei causes devastating and lethal diseases in humans and livestock. This parasite continuously evades the host adaptive immune response by drawing on a library of variant surface proteins but the mechanisms determining the timing of surface protein -host interactions are not understood. We report a simple mechanism, based on differential growth of parasites with surface proteins of different lengths, which can explain the hierarchy of variants over time. This allows parasites to evade host immune responses for extended timeframes using limited cohorts of surface proteins. We liken this strategy to a military 'feint attack', that enhances the parasites ability to evade the host immune response. A similar mechanism may also operate in other important pathogens.

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Depletion of the Trypanosome Pumilio Domain Protein PUF2 or of Some Other Essential Proteins Causes Transcriptome Changes Related to Coding Region Length

Cited by 24 publications

References 73 publications

Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA‐binding proteome

Gene expression regulatory networks in Trypanosoma brucei: insights into the role of the mRNA‐binding proteome

Expression of normal levels of the Pumilio domain protein PUF3 is required for optimal growth of Trypanosoma brucei

Faster growth with shorter antigens explains a VSG hierarchy during African trypanosome infections: a feint attack by parasites

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