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mRNA interactome capture in mammalian cells

Nicolai Kastelic, Markus Landthaler
2017 Methods  
Landthaler, mRNA interactome capture in mammalian cells, Methods (2017), doi: http://dx.  ... 
doi:10.1016/j.ymeth.2017.07.006 pmid:28710009 fatcat:n36kqmy7bfguroeyhuwub4bz7m

High-resolution profiling of protein occupancy on polyadenylated RNA transcripts

Mathias Munschauer, Markus Schueler, Christoph Dieterich, Markus Landthaler
2014 Methods  
A key prerequisite to understand how gene regulatory processes are controlled by the interplay of RNAbinding proteins and ribonucleoprotein complexes with RNAs is the generation of comprehensive highresolution maps of protein-RNA interactions. Recent advances in next-generation sequencing technology accelerated the development of various crosslinking and immunoprecipitation (CLIP) approaches to broadly identify RNA regions contacted by RNA-binding proteins. However these methods only consider
more » ... ngle RNA-binding proteins and their contact sites, irrespective of the overall cis-regulatory sequence space contacted by other RNA interacting factors. Here we describe the application of protein occupancy profiling, a novel approach that globally displays the RNA contact sites of the poly(A)+ RNA-bound proteome. Protein occupancy profiling enables the generation of transcriptome-wide maps of protein-RNA interactions on polyadenylated transcripts and narrows the sequence search space for transcript regions involved in cis-regulation of gene expression in response to internal or external stimuli, altered cellular programs or disease.
doi:10.1016/j.ymeth.2013.09.017 pmid:24096003 fatcat:k5yjyivyxrdqzezwyqq3wemqpi

Differential protein occupancy profiling of the mRNA transcriptome

Markus Schueler, Mathias Munschauer, Lea Haarup Gregersen, Ana Finzel, Alexander Loewer, Wei Chen, Markus Landthaler, Christoph Dieterich
2014 Genome Biology  
Acknowledgements We would like to thank Emanuel Wyler from the Landthaler lab for helpful discussions regarding the finding of differential occupancy positions.  ... 
doi:10.1186/gb-2014-15-1-r15 pmid:24417896 pmcid:PMC4056462 fatcat:oaozg7axnvebhmcis4jpjnj3zu

ENGINEERING, DECODING AND SYSTEMS-LEVEL CHARACTERIZATION OF CHIMPANZEE CYTOMEGALOVIRUS [article]

Quang Vinh Phan, Boris Bogdanow, Emanuel Wyler, Markus Landthaler, Fan Liu, Christian Hagemeier, Lueder Wiebusch
2021 bioRxiv   pre-print
The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious
more » ... iruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the transcriptome and proteome of infected cells and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.
doi:10.1101/2021.07.20.453063 fatcat:gfdad2qtqzet7j2vc7zjugbyqe

Identification of Novel Argonaute-Associated Proteins

Gunter Meister, Markus Landthaler, Lasse Peters, Po Yu Chen, Henning Urlaub, Reinhard Lührmann, Thomas Tuschl
2005 Current Biology  
RNA silencing processes are guided by small RNAs known as siRNAs and microRNAs (miRNAs) [1] [2] [3] [4] . They reside in ribonucleoprotein complexes, which guide the cleavage of complementary mRNAs [3, 4] or affect stability and translation of partial complementary mRNAs [1, 2, 5] . Argonaute (Ago) proteins are at the heart of silencing effector complexes and bind the single-stranded siRNA and miRNA [4, 6] . Our biochemical analysis revealed that Ago2 is present in a pre-miRNA processing
more » ... that is able to transfer the miRNA into a target-mRNA cleaving complex. To gain insight into the function and composition of RNA silencing complexes, we purified Ago1-and Ago2containing complexes from human cells. Several known Ago1-and/or Ago2-associated proteins including Dicer were identified, but also two novel factors, the putative RNA helicase MOV10, and the RNA recognition motif (RRM)-containing protein TNRC6B/ KIAA1093. The new proteins localize, similar to Ago proteins, to mRNA-degrading cytoplasmic P bodies, and they are functionally required to mediate miRNAguided mRNA cleavage. Results and Discussion Proteomic Analysis of Human Ago1 and Ago2 Complexes We generated HeLa-cell lines stably expressing FLAG/ HA-tagged Ago1 or Ago2 [7] and biochemically copurified Ago-associated proteins from HeLa-cell cytoplasmic extracts by double-affinity purification. Cytoplasmic extract was first passed over anti-FLAG-antibodycoated beads. Bound proteins were eluted under native
doi:10.1016/j.cub.2005.10.048 pmid:16289642 fatcat:3ryxfxq55rdqppvbuhv2bkkeki

Intense pulsed light (IPL): A review

Philipp Babilas, Stephan Schreml, Rolf-Markus Szeimies, Michael Landthaler
2010 Lasers in Surgery and Medicine  
Intense pulsed light (IPL) devices use flashlamps and bandpass filters to emit polychromatic incoherent high-intensity pulsed light of determined wavelength spectrum, fluence, and pulse duration. Similar to lasers, the basic principle of IPL devices is a more or less selective thermal damage of the target. The combination of prescribed wavelengths, fluences, pulse durations, and pulse intervals facilitates the treatment of a wide spectrum of skin conditions. Objective: To summarize the physics
more » ... f IPL, to provide guidance for the practical use of IPL devices, and to discuss the current literature on IPL in the treatment of unwanted hair growth, vascular lesions, pigmented lesions, acne vulgaris, and photodamaged skin and as a light source for PDT and skin rejuvenation. Methods: A systematic search of several electronic databases, including Medline and PubMed and the authors experience on intense pulsed light. Results: Numerous trials show the effectiveness and compatibility of IPL devices. Conclusion: Most comparative trials attest IPLs similar effectiveness to lasers (level of evidence: 2b to 4, depending on the indication). However, large controlled and blinded comparative trials with an extended follow-up period are necessary. Lasers Surg. Med. 42:93-104, 2010.
doi:10.1002/lsm.20877 pmid:20166155 fatcat:3avmu2rmvnhg7awlp23ghayvay

Spatio-temporal mRNA dynamics in the early zebrafish embryo [article]

Karoline Holler, Anika Neuschulz, Philipp Drewe-Bo&szlig, Janita Mintcheva, Bastiaan Spanjaard, Roberto Arsi&egrave, Uwe Ohler, Markus Landthaler, Jan Philipp Junker
2020 biorxiv/medrxiv   pre-print
Early stages of embryogenesis depend heavily on subcellular localization and transport of maternally deposited mRNA. However, systematic analysis of these processes is currently hindered by a lack of spatio-temporal information in single-cell RNA sequencing. Here, we combined spatially-resolved transcriptomics and single-cell RNA labeling to study the spatio-temporal dynamics of the transcriptome during the first few hours of zebrafish development. We measured spatial localization of mRNA
more » ... les with sub-single-cell resolution at the one-cell stage, which allowed us to identify a class of mRNAs that are specifically localized at an extraembryonic position, the vegetal pole. Furthermore, we established a method for high-throughput single-cell RNA labeling in early zebrafish embryos, which enabled us to follow the fate of individual maternal transcripts until gastrulation. This approach revealed that many localized transcripts are specifically transported to the primordial germ cells. Finally, we acquired spatial transcriptomes of two xenopus species, and we compared evolutionary conservation of localized genes as well as enriched sequence motifs. In summary, we established sub-single-cell spatial transcriptomics and single-cell RNA labeling to reveal principles of mRNA localization in early vertebrate development.
doi:10.1101/2020.11.19.389809 fatcat:4tkxdok2dfgddi7r7bl63x36am

DDX3 depletion selectively represses translation of structured mRNAs [article]

Lorenzo Calviello, Srivats Venkataramanan, Karol J. Rogowski, Emanuel Wyler, Malvika Tejura, Bao Thai, Jacek Krol, Witold Filipowicz, Markus Landthaler, Stephen N. Floor
2019 biorxiv/medrxiv   pre-print
AbstractDDX3 is an RNA chaperone of the DEAD-box family that regulates translation. Its yeast ortholog Ded1 controls the translation of nearly all mRNAs, whereas DDX3 is thought to regulate only a subset of mRNAs. However, the set of mRNAs that are regulated by DDX3 are unknown, along with the relationship between DDX3 binding and activity. Here, we use ribosome profiling, RNA-seq, and PAR-CLIP to define the set of mRNAs that are regulated by DDX3 in human cells. We find that while DDX3 binds
more » ... st expressed mRNAs, depletion of DDX3 affects the translation level of only a small subset of the transcriptome. We further find that DDX3 binds a site on helix 16 of the human ribosome, placing it immediately adjacent to the mRNA entry channel and translation factor eIF4B. Translation changes caused by depleting DDX3 levels or through chemical inhibition mimicking a dominant negative allele are different. Taken together, our data defines the subset of the transcriptome that is responsive to DDX3 inhibition, with relevance for basic biology and disease states where DDX3 expression is altered.
doi:10.1101/589218 fatcat:nhihgqf4bbfozdybvmqk2bkgru

In vitro Kinase-to-Phosphosite database (iKiP-DB) predicts kinase activity in phosphoproteomic datasets [article]

Tommaso Mari, Kristin Moesbauer, Emanuel Wyler, Markus Landthaler, Christian Drosten, Matthias Selbach
2022 bioRxiv   pre-print
Phosphoproteomics routinely quantifies changes in the levels of thousands of phosphorylation sites, but functional analysis of such data remains a major challenge. While databases like PhosphoSitePlus contain information about many phosphorylation sites, the vast majority of known sites are not assigned to any protein kinase. Assigning changes in the phosphoproteome to the activity of individual kinases therefore remains a key challenge.. A recent large-scale study systematically identified in
more » ... itro substrates for most human protein kinases. Here, we reprocessed and filtered these data to generate an in vitro Kinase-to-Phosphosite database (iKiP-DB). We show that iKiP-DB can accurately predict changes in kinase activity in published phosphoproteomic datasets for both well-studied and poorly characterized kinases. We apply iKiP-DB to a newly generated phosphoproteomic analysis of SARS-CoV-2 infected human lung epithelial cells and provide evidence for coronavirus-induced changes in host cell kinase activity. In summary, we show that iKiP-DB is widely applicable to facilitate the functional analysis of phosphoproteomic datasets.
doi:10.1101/2022.01.13.476159 fatcat:x7j4l3inrbf4fcu2pbznxapwcu

A Variety of Dicer Substrates in Human and C. elegans

Agnieszka Rybak-Wolf, Marvin Jens, Yasuhiro Murakawa, Margareta Herzog, Markus Landthaler, Nikolaus Rajewsky
2014 Cell  
The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/mi-croRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer-binding sites. We find known and hundreds of additional miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets.
more » ... most Dicer-binding sites reside on mRNAs/ lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside of the miRNA pathway.
doi:10.1016/j.cell.2014.10.040 pmid:25416952 fatcat:soletymivfcb3ky7sxb3n3ivna

Roquin binding to target mRNAs involves a winged helix-turn-helix motif

Anja Schuetz, Yasuhiro Murakawa, Eva Rosenbaum, Markus Landthaler, Udo Heinemann
2014 Nature Communications  
Roquin proteins mediate mRNA deadenylation by recognizing a conserved class of stem-loop RNA degradation motifs via their roquin domain. Here, we present the crystal structure of a roquin domain, and by combining structural, biochemical and mutation analyses we gain insight into the mode of RNA binding. We show that the winged helix-turn-helix motif is involved in the binding of constitutive decay elements-containing stem-loop mRNAs. Moreover, we provide biochemical evidence that roquin
more » ... are additionally able to bind to duplex RNA and have the potential to be functional in different oligomeric states.
doi:10.1038/ncomms6701 pmid:25504471 fatcat:g5thuw6qmnbt3mzvpuiow5l6nq

JACUSA: site-specific identification of RNA editing events from replicate sequencing data

Michael Piechotta, Emanuel Wyler, Uwe Ohler, Markus Landthaler, Christoph Dieterich
2017 BMC Bioinformatics  
Acknowledgments All authors would like thank all members of the Dieterich and Landthaler Labs for numerous valuable discussions. Funding  ... 
doi:10.1186/s12859-016-1432-8 pmid:28049429 pmcid:PMC5210316 fatcat:txo23tnmbvdeldlfmp7shnx33a

Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus

Quang Vinh Phan, Boris Bogdanow, Emanuel Wyler, Markus Landthaler, Fan Liu, Christian Hagemeier, Lüder Wiebusch, Daniel Malouli
2022 PLoS Pathogens  
The chimpanzee cytomegalovirus (CCMV) is the closest relative of human CMV (HCMV). Because of the high conservation between these two species and the ability of human cells to fully support CCMV replication, CCMV holds great potential as a model system for HCMV. To make the CCMV genome available for precise and rapid gene manipulation techniques, we captured the genomic DNA of CCMV strain Heberling as a bacterial artificial chromosome (BAC). Selected BAC clones were reconstituted to infectious
more » ... iruses, growing to similar high titers as parental CCMV. DNA sequencing confirmed the integrity of our clones and led to the identification of two polymorphic loci and a deletion-prone region within the CCMV genome. To re-evaluate the CCMV coding potential, we analyzed the viral transcriptome and proteome and identified several novel ORFs, splice variants, and regulatory RNAs. We further characterized the dynamics of CCMV gene expression and found that viral proteins cluster into five distinct temporal classes. In addition, our datasets revealed that the host response to CCMV infection and the de-regulation of cellular pathways are in line with known hallmarks of HCMV infection. In a first functional experiment, we investigated a proposed frameshift mutation in UL128 that was suspected to restrict CCMV's cell tropism. In fact, repair of this frameshift re-established productive CCMV infection in endothelial and epithelial cells, expanding the options of CCMV as an infection model. Thus, BAC-cloned CCMV can serve as a powerful tool for systematic approaches in comparative functional genomics, exploiting the close phylogenetic relationship between CCMV and HCMV.
doi:10.1371/journal.ppat.1010193 pmid:34982803 pmcid:PMC8759705 fatcat:letktvfsnrafbgp4eiqojwbkbe

Global analysis of human-to-mouse intercellular RNA transfer in cell culture [article]

Sandipan Dasgupta, Daniella Y. Dyagi, Gal Haimovich, Emanuel Wyler, Tsviya Olender, Robert H. Singer, Markus Landthaler, Jeffrey E Gerst
2021 bioRxiv   pre-print
Full-length mRNAs can transfer between adjacent mammalian cells via direct cell-to-cell connections called tunneling nanotubes (TNTs). However, the extent of mRNA transfer at the transcriptome-wide level (the transferome) is unknown. Here, we analyzed whole transcriptome mRNA transfer between heterogeneous human-mouse cell populations in in vitro co-culture using RNA-sequencing. Our data indicate that mRNA transfer is non-selective, prevalent across the human transcriptome, and that the amount
more » ... f transfer to mouse embryonic fibroblasts (MEFs) strongly correlates with the endogenous level of gene expression in donor human breast cancer cells (MCF7). These results were validated by both quantitative RT-PCR and in situ hybridization, and analysis shows that typically <1% of endogenous mRNAs and lncRNAs undergo transfer. Non-selective expression-dependent RNA transfer was further validated using synthetic RNA reporters. Notably, significant differential changes in the native MEF transcriptome were observed in response to co-culture, including the upregulation of multiple cancer- and cancer-associated fibroblast-related genes and pathways. Together, these results lead us to suggest that TNT-mediated RNA transfer could be a phenomenon of physiological importance under both normal and pathogenic conditions.
doi:10.1101/2021.11.28.470233 fatcat:rtlsoafksbhpliocdnusmrc5uq

DNA Binding and Cleavage by the HNH Homing Endonuclease I-HmuI

Betty W. Shen, Markus Landthaler, David A. Shub, Barry L. Stoddard
2004 Journal of Molecular Biology  
The structure of I-HmuI, which represents the last family of homing endonucleases without a defining crystallographic structure, has been determined in complex with its DNA target. A series of diverse protein structural domains and motifs, contacting sequential stretches of nucleotide bases, are distributed along the DNA target. I-HmuI contains an N-terminal domain with a DNA-binding surface found in the I-PpoI homing endonuclease and an associated HNH/N active site found in the bacterial
more » ... ns, and a C-terminal DNA-binding domain previously observed in the I-TevI homing endonuclease. The combination and exchange of these features between protein families indicates that the genetic mobility associated with homing endonucleases extends to the level of independent structural domains. I-HmuI provides an unambiguous structural connection between the His-Cys box endonucleases and the bacterial colicins, supporting the hypothesis that these enzymes diverged from a common ancestral nuclease.
doi:10.1016/j.jmb.2004.07.032 pmid:15313606 fatcat:g7nuclrejjgxlnw6juovyh55py
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