@article{KramerMeyerNatusStigloheretal.2021, author = {Kramer, Susanne and Meyer-Natus, Elisabeth and Stigloher, Christian and Thoma, Hanna and Schnaufer, Achim and Engstler, Markus}, title = {Parallel monitoring of RNA abundance, localization and compactness with correlative single molecule FISH on LR White embedded samples}, series = {Nucleic Acids Research}, volume = {49}, journal = {Nucleic Acids Research}, number = {3}, doi = {10.1093/nar/gkaa1142}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-230647}, year = {2021}, abstract = {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.}, language = {en} } @article{CastanedaLondonoBanholzerBannermannetal.2021, author = {Casta{\~n}eda Londono, Paula Andrea and Banholzer, Nicole and Bannermann, Bridget and Kramer, Susanne}, title = {Is mRNA decapping activity of ApaH like phosphatases (ALPH's) the reason for the loss of cytoplasmic ALPH's in all eukaryotes but Kinetoplastida?}, series = {BMC Ecology and Evolution}, volume = {21}, journal = {BMC Ecology and Evolution}, doi = {10.1186/s12862-021-01858-x}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261180}, year = {2021}, abstract = {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.}, language = {en} } @article{BakariSoaleIkengaScheibeetal.2021, author = {Bakari-Soale, Majeed and Ikenga, Nonso Josephat and Scheibe, Marion and Butter, Falk and Jones, Nicola G. and Kramer, Susanne and Engstler, Markus}, title = {The nucleolar DExD/H protein Hel66 is involved in ribosome biogenesis in Trypanosoma brucei}, series = {Scientific Reports}, volume = {11}, journal = {Scientific Reports}, number = {1}, doi = {10.1038/s41598-021-97020-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-263872}, year = {2021}, abstract = {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.}, language = {en} }