TY - JOUR A1 - Kramer, Susanne T1 - Simultaneous detection of mRNA transcription and decay intermediates by dual colour single mRNA FISH on subcellular resolution JF - Nucleic Acids Research N2 - The detection of mRNAs undergoing transcription or decay is challenging, because both processes are fast. However, the relative proportion of an mRNA in synthesis or decay increases with mRNA size and decreases with mRNA half-life. Based on this rationale, I have exploited a 22 200 nucleotide-long, short-lived endogenous mRNA as a reporter for mRNA metabolism in trypanosomes. The extreme 5΄ and 3΄ ends were labeled with red- and green-fluorescent Affymetrix® single mRNA FISH probes, respectively. In the resulting fluorescence images, yellow spots represent intact mRNAs; red spots are mRNAs in transcription or 3΄-5΄ decay, and green spots are mRNAs in 5΄-3΄ degradation. Most red spots were nuclear and insensitive to transcriptional inhibition and thus likely transcription intermediates. Most green spots were cytoplasmic, confirming that the majority of cytoplasmic decay in trypanosomes is 5΄-3΄. The system showed the expected changes at inhibition of transcription or translation and RNAi depletion of the trypanosome homologue to the 5΄-3΄ exoribonuclease Xrn1. The method allows to monitor changes in mRNA metabolism both on cellular and on population/tissue wide levels, but also to study the subcellular localization of mRNA transcription and decay pathways. I show that the system is applicable to mammalian cells. KW - mRNA Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148002 ER - TY - JOUR A1 - Fritz, Melanie A1 - Vanselow, Jens A1 - Sauer, Nadja A1 - Lamer, Stephanie A1 - Goos, Carina A1 - Siegel, T. Nicolai A1 - Subota, Ines A1 - Schlosser, Andreas A1 - Carrington, Mark A1 - Kramer, Susanne T1 - Novel insights into RNP granules by employing the trypanosome's microtubule skeleton as a molecular sieve JF - Nucleic Acids Research N2 - 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. Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-126180 ER - TY - JOUR A1 - Castañeda Londono, Paula Andrea A1 - Banholzer, Nicole A1 - Bannermann, Bridget A1 - Kramer, Susanne T1 - Is mRNA decapping activity of ApaH like phosphatases (ALPH’s) the reason for the loss of cytoplasmic ALPH’s in all eukaryotes but Kinetoplastida? JF - BMC Ecology and Evolution N2 - Background: ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and are present in eukaryotes of all eukaryotic super-groups; still, only two proteins have been functionally characterised. One is ALPH1 from the Kinetoplastid Trypanosoma brucei that we recently found to be the mRNA decapping enzyme of the parasite. mRNA decapping by ALPHs is unprecedented in eukaryotes, which usually use nudix hydrolases, but the bacterial ancestor protein ApaH was recently found to decap non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or more widespread among eukaryotes. Results: We screened 824 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to refine phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of ALPHs. We found that most eukaryotes have either no ALPH (500/824) or very short ALPHs, consisting almost exclusively of the catalytic domain. These ALPHs had mostly predicted non-cytoplasmic localisations, often supported by the presence of transmembrane helices and signal peptides and in two cases (one in this study) by experimental data. The only exceptions were ALPH1 homologues from Kinetoplastida, that all have unique C-terminal and mostly unique N-terminal extension, and at least the T. brucei enzyme localises to the cytoplasm. Surprisingly, despite of these non-cytoplasmic localisations, ALPHs from all eukaryotic super-groups had in vitro mRNA decapping activity. Conclusions: ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme since, or use it exclusively outside the cytoplasm in organelles in a version consisting of the catalytic domain only. While our data provide no evidence for the presence of further mRNA decapping enzymes among eukaryotic ALPHs, the broad substrate range of ALPHs that includes mRNA caps provides an explanation for the selection against the presence of a cytoplasmic ALPH protein as a mean to protect mRNAs from unregulated degradation. Kinetoplastida succeeded to exploit ALPH as their mRNA decapping enzyme, likely using the Kinetoplastida-unique N- and C-terminal extensions for regulation. KW - ApaH like phosphatase KW - ApaH KW - ALPH KW - Trypanosoma brucei KW - mRNA decapping KW - m7G cap KW - mRNA cap KW - ALPH1 KW - Kinetoplastida Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261180 VL - 21 ER - TY - JOUR A1 - Kramer, Susanne A1 - Piper, Sophie A1 - Estevez, Antonio A1 - Carrington, Mark T1 - Polycistronic trypanosome mRNAs are a target for the exosome JF - Molecular and Biochemical Parasitology N2 - Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNA5 from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5'-3' exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNA5. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. KW - Trypanosoma brucei KW - Exosome KW - NMD KW - Polycistronic mRNA KW - trans-splicing KW - Trypanosomes Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-191350 VL - 205 IS - 1-2 ER - TY - JOUR A1 - Kramer, Susanne A1 - Meyer-Natus, Elisabeth A1 - Stigloher, Christian A1 - Thoma, Hanna A1 - Schnaufer, Achim A1 - Engstler, Markus T1 - Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples JF - Nucleic Acids Research N2 - Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans. KW - mRNA Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-230647 VL - 49 IS - 3 ER - TY - JOUR A1 - Vellmer, Tim A1 - Hartleb, Laura A1 - Fradera Sola, Albert A1 - Kramer, Susanne A1 - Meyer-Natus, Elisabeth A1 - Butter, Falk A1 - Janzen, Christian J. T1 - A novel SNF2 ATPase complex in Trypanosoma brucei with a role in H2A.Z-mediated chromatin remodelling JF - PLoS Pathogens N2 - A cascade of histone acetylation events with subsequent incorporation of a histone H2A variant plays an essential part in transcription regulation in various model organisms. A key player in this cascade is the chromatin remodelling complex SWR1, which replaces the canonical histone H2A with its variant H2A.Z. Transcriptional regulation of polycistronic transcription units in the unicellular parasite Trypanosoma brucei has been shown to be highly dependent on acetylation of H2A.Z, which is mediated by the histone-acetyltransferase HAT2. The chromatin remodelling complex which mediates H2A.Z incorporation is not known and an SWR1 orthologue in trypanosomes has not yet been reported. In this study, we identified and characterised an SWR1-like remodeller complex in T. brucei that is responsible for Pol II-dependent transcriptional regulation. Bioinformatic analysis of potential SNF2 DEAD/Box helicases, the key component of SWR1 complexes, identified a 1211 amino acids-long protein that exhibits key structural characteristics of the SWR1 subfamily. Systematic protein-protein interaction analysis revealed the existence of a novel complex exhibiting key features of an SWR1-like chromatin remodeller. RNAi-mediated depletion of the ATPase subunit of this complex resulted in a significant reduction of H2A.Z incorporation at transcription start sites and a subsequent decrease of steady-state mRNA levels. Furthermore, depletion of SWR1 and RNA-polymerase II (Pol II) caused massive chromatin condensation. The potential function of several proteins associated with the SWR1-like complex and with HAT2, the key factor of H2A.Z incorporation, is discussed. KW - Trypanosoma KW - chromatin KW - histones KW - RNA interference KW - Trypanosoma brucei gambiense KW - luciferase KW - transcriptional control KW - nucleosomes Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-301372 VL - 18 IS - 6 ER - TY - JOUR A1 - Bakari-Soale, Majeed A1 - Ikenga, Nonso Josephat A1 - Scheibe, Marion A1 - Butter, Falk A1 - Jones, Nicola G. A1 - Kramer, Susanne A1 - Engstler, Markus T1 - The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei JF - Scientific Reports N2 - The biosynthesis of ribosomes is a complex cellular process involving ribosomal RNA, ribosomal proteins and several further trans-acting factors. DExD/H box proteins constitute the largest family of trans-acting protein factors involved in this process. Several members of this protein family have been directly implicated in ribosome biogenesis in yeast. In trypanosomes, ribosome biogenesis differs in several features from the process described in yeast. Here, we have identified the DExD/H box helicase Hel66 as being involved in ribosome biogenesis. The protein is unique to Kinetoplastida, localises to the nucleolus and its depletion via RNAi caused a severe growth defect. Loss of the protein resulted in a decrease of global translation and accumulation of rRNA processing intermediates for both the small and large ribosomal subunits. Only a few factors involved in trypanosome rRNA biogenesis have been described so far and our findings contribute to gaining a more comprehensive picture of this essential process. KW - infection KW - parasite evolution KW - parasite genetics KW - RNA Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-263872 VL - 11 IS - 1 ER - TY - JOUR A1 - Zoltner, Martin A1 - Krienitz, Nina A1 - Field, Mark C. A1 - Kramer, Susanne T1 - Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA JF - PLoS Neglected Tropical Diseases N2 - Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes. KW - Trypanosoma KW - mRNA KW - T. brucei KW - PABPs Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-177126 VL - 12 IS - 7 ER - TY - JOUR A1 - Kramer, Susanne T1 - The ApaH-like phosphatase TbALPH1 is the major mRNA decapping enzyme of trypanosomes JF - PLoS Pathogens N2 - 5’-3’ decay is the major mRNA decay pathway in many eukaryotes, including trypanosomes. After deadenylation, mRNAs are decapped by the nudix hydrolase DCP2 of the decapping complex and finally degraded by the 5’-3’ exoribonuclease. Uniquely, trypanosomes lack homologues to all subunits of the decapping complex, while deadenylation and 5’-3’ degradation are conserved. Here, I show that the parasites use an ApaH-like phosphatase (ALPH1) as their major mRNA decapping enzyme. The protein was recently identified as a novel trypanosome stress granule protein and as involved in mRNA binding. A fraction of ALPH1 co-localises exclusively with the trypanosome 5’-3’ exoribonuclease XRNA to a special granule at the posterior pole of the cell, indicating a connection between the two enzymes. RNAi depletion of ALPH1 is lethal and causes a massive increase in total mRNAs that are deadenylated, but have not yet started 5’-3’ decay. These data suggest that ALPH1 acts downstream of deadenylation and upstream of mRNA degradation, consistent with a function in mRNA decapping. In vitro experiments show that recombinant, N-terminally truncated ALHP1 protein, but not a catalytically inactive mutant, sensitises the capped trypanosome spliced leader RNA to yeast Xrn1, but only if an RNA 5’ polyphosphatase is included. This indicates that the decapping mechanism of ALPH1 differs from the decapping mechanism of Dcp2 by leaving more than one phosphate group at the mRNA’s 5’ end. This is the first reported function of a eukaryotic ApaH-like phosphatase, a bacterial-derived class of enzymes present in all phylogenetic super-groups of the eukaryotic kingdom. The substrates of eukaryotic ApaH-like phosphatases are unknown. However, the substrate of the related bacterial enzyme ApaH, diadenosine tetraphosphate, is highly reminiscent of a eukaryotic mRNA cap. KW - eukaryota KW - Trypanosoma KW - RNA interference KW - messenger RNA Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158482 VL - 13 IS - 6 ER - TY - JOUR A1 - Goos, Carina A1 - Dejung, Mario A1 - Janzen, Christian J. A1 - Butter, Falk A1 - Kramer, Susanne T1 - The nuclear proteome of Trypanosoma brucei JF - PLoS ONE N2 - Trypanosoma brucei is a protozoan flagellate that is transmitted by tsetse flies into the mammalian bloodstream. The parasite has a huge impact on human health both directly by causing African sleeping sickness and indirectly, by infecting domestic cattle. The biology of trypanosomes involves some highly unusual, nuclear-localised processes. These include polycistronic transcription without classical promoters initiated from regions defined by histone variants, trans-splicing of all transcripts to the exon of a spliced leader RNA, transcription of some very abundant proteins by RNA polymerase I and antigenic variation, a switch in expression of the cell surface protein variants that allows the parasite to resist the immune system of its mammalian host. Here, we provide the nuclear proteome of procyclic Trypanosoma brucei, the stage that resides within the tsetse fly midgut. We have performed quantitative label-free mass spectrometry to score 764 significantly nuclear enriched proteins in comparison to whole cell lysates. A comparison with proteomes of several experimentally characterised nuclear and non-nuclear structures and pathways confirmed the high quality of the dataset: the proteome contains about 80% of all nuclear proteins and less than 2% false positives. Using motif enrichment, we found the amino acid sequence KRxR present in a large number of nuclear proteins. KRxR is a sub-motif of a classical eukaryotic monopartite nuclear localisation signal and could be responsible for nuclear localization of proteins in Kinetoplastida species. As a proof of principle, we have confirmed the nuclear localisation of six proteins with previously unknown localisation by expressing eYFP fusion proteins. While proteome data of several T. brucei organelles have been published, our nuclear proteome closes an important gap in knowledge to study trypanosome biology, in particular nuclear-related processes. KW - Trypanosoma KW - gambiense KW - Trypanosoma brucei KW - proteomes KW - yellow fluorescent protein KW - mitochondria KW - protein structure KW - histones Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-158572 VL - 12 IS - 7 ER -