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Bartoschek, M.D., Ugur, E., Nguyen, T.A., Rodschinka, G., Wierer, M., Lang, K., and Bultmann, S.
(IMPRS-LS students are in bold)
Nucleic Acids Res, 2021, online ahead of print.
doi: 10.1093/nar/gkab132

Identification of permissive amber suppression sites for efficient non-canonical amino acid incorporation in mammalian cells

The genetic code of mammalian cells can be expanded to allow the incorporation of non-canonical amino acids (ncAAs) by suppressing in-frame amber stop codons (UAG) with an orthogonal pyrrolysyl-tRNA synthetase (PylRS)/tRNAPylCUA (PylT) pair. However, the feasibility of this approach is substantially hampered by unpredictable variations in incorporation efficiencies at different stop codon positions within target proteins. Here, we apply a proteomics-based approach to quantify ncAA incorporation rates at hundreds of endogenous amber stop codons in mammalian cells. With these data, we compute iPASS (Identification of Permissive Amber Sites for Suppression; available at, a linear regression model to predict relative ncAA incorporation efficiencies depending on the surrounding sequence context. To verify iPASS, we develop a dual-fluorescence reporter for high-throughput flow-cytometry analysis that reproducibly yields context-specific ncAA incorporation efficiencies. We show that nucleotides up- and downstream of UAG synergistically influence ncAA incorporation efficiency independent of cell line and ncAA identity. Additionally, we demonstrate iPASS-guided optimization of ncAA incorporation rates by synonymous exchange of codons flanking the amber stop codon. This combination of in silico analysis followed by validation in living mammalian cells substantially simplifies identification as well as adaptation of sites within a target protein to confer high ncAA incorporation rates.



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Peritore, M., Reusswig, K.U., Bantele, S.C.S., Straub, T., and Pfander, B.
Mol Cell, 2021, online ahead of print.
doi: 10.1016/j.molcel.2021.02.005

Strand-specific ChIP-seq at DNA breaks distinguishes ssDNA versus dsDNA binding and refutes single-stranded nucleosomes

In a first step of DNA double-strand break (DSB) repair by homologous recombination, DNA ends are resected such that single-stranded DNA (ssDNA) overhangs are generated. ssDNA is specifically bound by RPA and other factors, which constitutes a ssDNA-domain on damaged chromatin. The molecular organization of this ssDNA and the adjacent dsDNA domain is crucial during DSB signaling and repair. However, data regarding the presence of nucleosomes, the most basic chromatin components, in the ssDNA domain have been contradictory. Here, we use site-specific induction of DSBs and chromatin immunoprecipitation followed by strand-specific sequencing to analyze in vivo binding of key DSB repair and signaling proteins to either the ssDNA or dsDNA domain. In the case of nucleosomes, we show that recently proposed ssDNA nucleosomes are not a major, persistent species, but that nucleosome eviction and DNA end resection are intrinsically coupled. These results support a model of separated dsDNA-nucleosome and ssDNA-RPA domains during DSB repair.



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Zeitler, L., Fiore, A., Meyer, C., Russier, M., Zanella, G., Suppmann, S., Gagaro, M., Sidhu, S.S., Seshagiri, S., Ohnmacht, C., Köcher, T., Fallarino, F., Linkermann, A., and Murray, P.J.
Elife, 2021, 10, online ahead of print.
doi: 10.7554/eLife.64806

Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologues in snake venoms (LAAO, L-amino acid oxidases), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell productive gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.



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Mengoli, V., Jonak, K., Lyzak, O., Lamb, M., Lister, L.M., Lodge, C., Rojas, J., Zagoriy, I., Herbert, M., and Zachariae, W.
(IMPRS-LS students are in bold)
EMBO J, 2021, e106812, online ahead of print.
doi: 10.15252/embj.2020106812

Deprotection of centromeric cohesin at meiosis II requires APC/C activity but not kinetochore tension

Genome haploidization involves sequential loss of cohesin from chromosome arms and centromeres during two meiotic divisions. At centromeres, cohesin's Rec8 subunit is protected from separase cleavage at meiosis I and then deprotected to allow its cleavage at meiosis II. Protection of centromeric cohesin by shugoshin-PP2A seems evolutionarily conserved. However, deprotection has been proposed to rely on spindle forces separating the Rec8 protector from cohesin at metaphase II in mammalian oocytes and on APC/C-dependent destruction of the protector at anaphase II in yeast. Here, we have activated APC/C in the absence of sister kinetochore biorientation at meiosis II in yeast and mouse oocytes, and find that bipolar spindle forces are dispensable for sister centromere separation in both systems. Furthermore, we show that at least in yeast, protection of Rec8 by shugoshin and inhibition of separase by securin are both required for the stability of centromeric cohesin at metaphase II. Our data imply that related mechanisms preserve the integrity of dyad chromosomes during the short metaphase II of yeast and the prolonged metaphase II arrest of mammalian oocytes.



Researchers at the Max Planck Institute of Biochemistry have established a new technique based on Next Generation Sequencing that determines whether a protein binds to single-stranded or double-stranded DNA.

DNA in our cells is constantly exposed to damaging agents, such as UV light or byproducts of cellular metabolism like reactive oxygen species. The most severe form of DNA damage are DNA double stranded breaks. In order to keep the genetic information intact, cells need to repair these double stranded breaks. The most faithful repair process is homologous recombination, which involves DNA end resection, the degradation of one DNA strand at each side of the break. Two different domains are therefore created around the double strand breaks: one containing single-stranded DNA and one containing double-stranded. Researchers from the team of Boris Pfander, head of the research group "DNA Replication and Genome Integrity" at the Max Planck Institute of Biochemistry have now developed a ChIP-seq-technique, based on chromatin-immunoprecipitation followed by next-generation sequencing.

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Congratulations on your PhD!


Hamid Hamzeiy

Advancing Computational Methods for Mass Spectrometry-Based Proteomics, Metabolomics, and Analysis of Multi-Omics Datasets

RG: Jürgen Cox



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Delgado de la Herran, H.C., Cheng, Y., and Perocchi, F.
Cell Calcium, 2021, 95, 102364.
doi: 10.1016/j.ceca.2021.102364

Towards a systems-level understanding of mitochondrial biology

Human mitochondria are complex and highly dynamic biological systems, comprised of over a thousand parts and evolved to fully integrate into the specialized intracellular signaling networks and metabolic requirements of each cell and organ. Over the last two decades, several complementary, top-down computational and experimental approaches have been developed to identify, characterize and modulate the human mitochondrial system, demonstrating the power of integrating classical reductionist and discovery-driven analyses in order to de-orphanize hitherto unknown molecular components of mitochondrial machineries and pathways. To this goal, systematic, multiomics-based surveys of proteome composition, protein networks, and phenotype-to-pathway associations at the tissue, cell and organellar level have been largely exploited to predict the full complement of mitochondrial proteins and their functional interactions, therefore catalyzing data-driven hypotheses. Collectively, these multidisciplinary and integrative research approaches hold the potential to propel our understanding of mitochondrial biology and provide a systems-level framework to unraveling mitochondria-mediated and disease-spanning pathomechanisms.



Being constantly flooded by a mass of stimuli, it is impossible for us to react to all of them. The same holds true for a little fish. Which stimuli should it pay attention to and which not? Scientists at the Max Planck Institute of Neurobiology have now deciphered the neuronal circuit that zebrafish use to prioritize visual stimuli. Surrounded by predators, a fish can thus choose its escape route from this predicament.

Even though we are not exposed to predators, we still have to decide which stimuli we pay attention to – for example, when crossing a street. Which cars should we avoid, which ones can we ignore? "The processes in the brain and the circuits that lead to this so-called selective attention are largely unexplored," explains Miguel Fernandes, a postdoctoral researcher in Herwig Baier's department. "But if we understand this in a simple animal model like the zebrafish, it can give us fundamental insights into decision-making mechanisms in humans." For this reason, Miguel Fernandes and his colleagues studied the behavior of zebrafish in the predicament described above: Using virtual reality, the team simulated two predators approaching a fish from the left and right at the same speed. In most cases, the fish focused on one of the two predators and fled in the opposite direction. They thus integrated only one, the so-called "winner stimulus", into their escape route (winner-take-all strategy).

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Congratulations on your PhD!


Stephan Holtkamp

Time-of-Day Dependent Trafficking of Leukocytes Across Lymphatics

RG: Christoph Scheiermann



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Behrens, A., Rodschinka, G., and Nedialkova, D.D.
(IMPRS-LS students in bold)
Mol Cell, 2021, online ahead of print.
doi: 10.1016/j.molcel.2021.01.028

High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq

Measurements of cellular tRNA abundance are hampered by pervasive blocks to cDNA synthesis at modified nucleosides and the extensive similarity among tRNA genes. We overcome these limitations with modification-induced misincorporation tRNA sequencing (mim-tRNAseq), which combines a workflow for full-length cDNA library construction from endogenously modified tRNA with a comprehensive and user-friendly computational analysis toolkit. Our method accurately captures tRNA abundance and modification status in yeast, fly, and human cells and is applicable to any organism with a known genome. We applied mim-tRNAseq to discover a dramatic heterogeneity of tRNA isodecoder pools among diverse human cell lines and a surprising interdependence of modifications at distinct sites within the same tRNA transcript.