Susanne Kramer

Heat shock causes a decrease in polysomes and the appearance of stress granules in trypanosomes independently of eIF2α phosphorylation at Thr169

In trypanosomes there is an almost total reliance on post-transcriptional mechanisms to alter gene expression; here, heat shock was used to investigate the response to an environmental signal. Heat shock rapidly and reversibly induced a decrease in polysome abundance, and the consequent changes in mRNA metabolism were studied. Both heat shock and polysome dissociation were necessary for (1) a reduction in mRNA levels that was more rapid than normal turnover, (2) an increased number of P-body-like granules that contained DHH1, SCD6 and XRNA, (3) the formation of stress granules that remained largely separate from the P-body-like granules and localise to the periphery of the cell and, (4) an increase in the size of a novel focus located at the posterior pole of the cell that contain XRNA, but neither DHH1 nor SCD6. The response differed from mammalian cells in that neither the decrease in polysomes nor stress-granule formation required phosphorylation of eIF2α at the position homologous to that of serine 51 in mammalian eIF2α and in the occurrence of a novel XRNA-focus.

Developmental regulation of gene expression in the absence of transcriptional control: The case of kinetoplastids

Kinetoplastids, including the human pathogens Trypanosoma brucei, Trypanosoma cruzi and Leishmania, are the only known organisms that do not regulate the transcription of protein coding genes transcribed by RNA polymerase II. Yet, profound changes in gene expression are induced by many different external stimuli and stresses, the extreme example are cascades of changes in gene expression initiated by differentiation triggers that ultimately and irreversibly result in the massive morphological and metabolic changes observed during life-cycle progression. This review explores how kinetoplastids change gene expression by looking at life-cycle stage specific changes in chromatin, mRNA processing, mRNA stability, mRNA translation, protein stability and protein modifications.

IL-2 and autoimmune disease.

A decade after the first description of IL-2-deficient mice, the redundancy of IL-2 as a T cell growth factor is well accepted and the focus of research has shifted to the unexpected multiorgan autoimmunity and inflammation observed in mice lacking components of the IL-2/IL-2R system. So far, a set of defects at the levels of repertoire selection, the generation of suppressive regulatory T cells, T cell homing and clonal contraction via activation induced cell death (AICD) have been documented. We propose that these individual defects jointly contribute to the severe disturbance of T cell homeostasis and self-tolerance underlying the immunopathology of the IL-2 deficiency syndrome.

Trans-acting proteins regulating mRNA maturation, stability and translation in trypanosomatids

In trypanosomatids, alterations in gene expression in response to intrinsic or extrinsic signals are achieved through post-transcriptional mechanisms. In the last 20 years, research has concentrated on defining the responsible cis-elements in the untranslated regions of several regulated mRNAs. More recently, the focus has shifted towards the identification of RNA-binding proteins that act as trans-acting factors. Trypanosomatids have a large number of predicted RNA-binding proteins of which the vast majority have no orthologues in other eukaryotes. Several RNA-binding proteins have been shown to bind and/or regulate the expression of a group of mRNAs that code for functionally related proteins, indicating the possible presence of co-regulated mRNA cohorts.

The RNA helicase DHH1 is central to the correct expression of many developmentally regulated mRNAs in trypanosomes

In trypanosomes, the predominant mechanisms of regulation of gene expression are post-transcriptional. The DEAD-box RNA helicase DHH1 was identified in a screen for gene products that are necessary for the instability of the GPI-PLC mRNA in insect-stage trypanosomes. Expression of an ATPase-deficient dhh1 mutant caused a rapid growth arrest associated with a decrease in polysomes, an increase in P-bodies and a slight decrease in average mRNA levels. However, the effect of dhh1 mutant expression on both turnover and translational repression of mRNAs was selective. Whereas there was little effect on the stability of constitutive mRNAs, the control of a large cohort of developmentally regulated mRNAs was reversed; many mRNAs normally downregulated in insect-stage trypanosomes were stabilized and many mRNAs normally upregulated decreased in level. One stabilised mRNA, ISG75, was characterised further. Despite the overall decrease in polysomes, the proportion of the ISG75 mRNA in polysomes was unchanged and the result was ISG75 protein accumulation. Our data show that specific mRNAs can escape DHH1-mediated translational repression. In trypanosomes, DHH1 has a selective role in determining the levels of developmentally regulated mRNAs.

HIV-1 Nef upregulates CCL2/MCP-1 expression in astrocytes in a myristoylation- and calmodulin-dependent manner

HIV-associated dementia (HAD) correlates with infiltration of monocytes into the brain. The accessory HIV-1 negative factor (Nef) protein, which modulates several signaling pathways, is constitutively present in persistently infected astroctyes. We demonstrated that monocytes responded with chemotaxis when subjected to cell culture supernatants of nef-expressing astrocytic U251MG cells. Using a protein array, we identified CC chemokine ligand 2/monocyte chemotactic protein-1 (CCL2/MCP-1) as a potential chemotactic factor mediating this phenomenon. CCL2/MCP-1 upregulation by Nef was further confirmed by ribonuclease protection assay, RT-PCR and ELISA. By applying neutralizing antibodies against CCL2/MCP-1 and using CCR2-deficient monocytes, we confirmed CCL2/MCP-1 as the exclusive factor secreted by nef-expressing astrocytes capable of attracting monocytes. Additionally, we showed that Nef-induced CCL2/MCP-1 expression depends on the myristoylation moiety of Nef and requires functional calmodulin. In summary, we suggest that Nef-induced CCL2/MCP-1 expression in astrocytes contributes to infiltration of monocytes into the brain, and thereby to progression of HAD.

Genome organization is a major component of gene expression control in response to stress and during the cell division cycle in trypanosomes.

The trypanosome genome is characterized by RNA polymerase II-driven polycistronic transcription of protein-coding genes. Ten to hundreds of genes are co-transcribed from a single promoter; thus, selective regulation of individual genes via initiation is impossible. However, selective responses to external stimuli occur and post-transcriptional mechanisms are thought to account for all temporal gene expression patterns. We show that genes encoding mRNAs that are differentially regulated during the heat-shock response are selectively positioned in polycistronic transcription units; downregulated genes are close to transcription initiation sites and upregulated genes are distant. We demonstrate that the position of a reporter gene within a transcription unit is sufficient to reproduce this effect. Analysis of gene ontology annotations reveals that positional bias is not restricted to stress–response genes and that there is a genome-wide organization based on proximity to transcription initiation sites. Furthermore, we show that the relative abundance of mRNAs at different time points in the cell division cycle is dependent on the location of the corresponding genes to transcription initiation sites. This work provides evidence that the genome in trypanosomes is organized to facilitate co-coordinated temporal control of gene expression in the absence of selective promoters.

Blocking Variant Surface Glycoprotein Synthesis in Trypanosoma brucei Triggers a General Arrest in Translation Initiation

Background The African trypanosome Trypanosoma brucei is covered with a dense layer of Variant Surface Glycoprotein (VSG), which protects it from lysis by host complement via the alternative pathway in the mammalian bloodstream. Blocking VSG synthesis by the induction of VSG RNAi triggers an unusually precise precytokinesis cell-cycle arrest. Methodology/Principal Findings Here, we characterise the cells arrested after the induction of VSG RNAi. We were able to rescue the VSG221 RNAi induced cell-cycle arrest through expression of a second different VSG (VSG117 which is not recognised by the VSG221 RNAi) from the VSG221 expression site. Metabolic labeling of the arrested cells showed that blocking VSG synthesis triggered a global translation arrest, with total protein synthesis reduced to less than 1–4% normal levels within 24 hours of induction of VSG RNAi. Analysis by electron microscopy showed that the translation arrest was coupled with rapid disassociation of ribosomes from the endoplasmic reticulum. Polysome analysis showed a drastic decrease in polysomes in the arrested cells. No major changes were found in levels of transcription, total RNA transcript levels or global amino acid concentrations in the arrested cells. Conclusions The cell-cycle arrest phenotype triggered by the induction of VSG221 RNAi is not caused by siRNA toxicity, as this arrest can be alleviated if a second different VSG is inserted downstream of the active VSG221 expression site promoter. Analysis of polysomes in the stalled cells showed that the translation arrest is mediated at the level of translation initiation rather than elongation. The cell-cycle arrest induced in the presence of a VSG synthesis block is reversible, suggesting that VSG synthesis and/or trafficking to the cell surface could be monitored during the cell-cycle as part of a specific cell-cycle checkpoint.

Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.

RNA in development: how ribonucleoprotein granules regulate the life cycles of pathogenic protozoa

Ribonucleoprotein (RNP) granules are important posttranscriptional regulators of messenger RNA (mRNA) fate. Several types of RNP granules specifically regulate gene expression during development of multicellular organisms and are commonly referred to as germ granules. The function of germ granules is not entirely understood and probably diverse, but it is generally agreed that one main function is posttranscriptional regulation of gene expression during early development, when transcription is silent. One example is the translational repression of maternally derived mRNAs in oocytes. Here, I hope to show that the need for regulation of gene expression by RNP granules is not restricted to animal development, but plays an equally important role during the development of pathogenic protozoa. Apicomplexa and Trypanosomatidae have complex life cycles with frequent host changes. The need to quickly adapt gene expression to a new environment as well as the ability to suppress translation to survive latencies is critical for successful completion of life cycles. Posttranscriptional gene regulation is not necessarily simpler in protozoa. Apicomplexa surprise with the presence of micro RNA (miRNAs) and upstream open reading frames (µORFs). Trypanosomes have an unusually large repertoire of different RNP granule types. A better understanding of RNP granules in protozoa may help to gain insight into the evolutionary origin of RNP granules: Trypanosomes for example have two types of granules with interesting similarities to animal germ granules. WIREs RNA 2014, 5:263–284. doi: 10.1002/wrna.1207