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  - 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  -