Publications by authors named "Matthias Meurer"

25 Publications

  • Page 1 of 1

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): study protocol for a two-factorial randomized controlled multi-arm trial with cluster sampling.

Trials 2021 Sep 26;22(1):656. Epub 2021 Sep 26.

Division of Clinical Tropical Medicine, University of Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.

Background: To achieve higher effectiveness in population-based SARS-CoV-2 surveillance and to reliably predict the course of an outbreak, screening, and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic.

Methods: This trial implements a two-factorial, randomized, controlled, multi-arm, prospective, interventional, single-blinded design with cluster sampling and four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy based on individuals' self-collection of saliva samples which are then sent to and analyzed by a laboratory. The targeted sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (2500 participants per arm). Strategies differ with respect to tested population groups (individuals vs. all household members) and testing approach (without vs. with pre-screening survey). The trial is complemented by an economic evaluation and qualitative assessment of user experiences. Primary outcomes include costs per completely screened person, costs per positive case, positive detection rate, and precision of positive detection rate.

Discussion: Systems for active surveillance of the general population will gain more importance in the context of pandemics and related disease prevention efforts. The pandemic parameters derived from such active surveillance with routine population monitoring therefore not only enable a prospective assessment of the short-term course of a pandemic, but also a more targeted and thus more effective use of local and short-term countermeasures.

Trial Registration: ClinicalTrials.gov DRKS00023271 . Registered November 30, 2020, with the German Clinical Trials Register (Deutsches Register Klinischer Studien).
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http://dx.doi.org/10.1186/s13063-021-05619-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8474710PMC
September 2021

Timer-based proteomic profiling of the ubiquitin-proteasome system reveals a substrate receptor of the GID ubiquitin ligase.

Mol Cell 2021 06 10;81(11):2460-2476.e11. Epub 2021 May 10.

Institute of Molecular Biology (IMB), Mainz, Germany. Electronic address:

Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
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http://dx.doi.org/10.1016/j.molcel.2021.04.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189435PMC
June 2021

Colorimetric RT-LAMP and LAMP-sequencing forDetecting SARS-CoV-2 RNA in Clinical Samples.

Bio Protoc 2021 Mar 20;11(6):e3964. Epub 2021 Mar 20.

Schaller Research Group, Department of Infectious Diseases, Virology, Heidelberg University, Heidelberg, Germany.

During pandemics, such as the one caused by SARS-CoV-2 coronavirus, simple methods to rapidly test large numbers of people are needed. As a faster and less resource-demanding alternative to detect viral RNA by conventional qPCR, we used reverse transcription loop-mediated isothermal amplification (RT-LAMP). We previously established colorimetric RT-LAMP assays on both purified and unpurified SARS-CoV-2 clinical specimens and further developed a multiplexed sequencing protocol (LAMP-sequencing) to analyze the outcome of many RT-LAMP reactions at the same time (Dao Thi , 2020). Extending on this work, we hereby provide step-by-step protocols for both RT-LAMP assays and read-outs.
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http://dx.doi.org/10.21769/BioProtoc.3964DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8032487PMC
March 2021

Effectiveness and cost-effectiveness of four different strategies for SARS-CoV-2 surveillance in the general population (CoV-Surv Study): a structured summary of a study protocol for a cluster-randomised, two-factorial controlled trial.

Trials 2021 Jan 8;22(1):39. Epub 2021 Jan 8.

Section Clinical Tropical Medicine, University of Heidelberg, Im Neuenheimer Feld 324, 69120, Heidelberg, Germany.

Objectives: In this cluster-randomised controlled study (CoV-Surv Study), four different "active" SARS-CoV-2 testing strategies for general population surveillance are evaluated for their effectiveness in determining and predicting the prevalence of SARS-CoV-2 infections in a given population. In addition, the costs and cost-effectiveness of the four surveillance strategies will be assessed. Further, this trial is supplemented by a qualitative component to determine the acceptability of each strategy. Findings will inform the choice of the most effective, acceptable and affordable strategy for SARS-CoV-2 surveillance, with the most effective and cost-effective strategy becoming part of the local public health department's current routine health surveillance activities. Investigating its everyday performance will allow us to examine the strategy's applicability to real time prevalence prediction and the usefulness of the resulting information for local policy makers to implement countermeasures that effectively prevent future nationwide lockdowns. The authors would like to emphasize the importance and relevance of this study and its expected findings in the context of population-based disease surveillance, especially in respect to the current SARS-CoV-2 pandemic. In Germany, but also in many other countries, COVID-19 surveillance has so far largely relied on passive surveillance strategies that identify individuals with clinical symptoms, monitor those cases who then tested positive for the virus, followed by tracing of individuals in close contact to those positive cases. To achieve higher effectiveness in population surveillance and to reliably predict the course of an outbreak, screening and monitoring of infected individuals without major symptoms (about 40% of the population) will be necessary. While current testing capacities are also used to identify such asymptomatic cases, this rather passive approach is not suitable in generating reliable population-based estimates of the prevalence of asymptomatic carriers to allow any dependable predictions on the course of the pandemic. To better control and manage the SARS-CoV-2 pandemic, current strategies therefore need to be complemented by an active surveillance of the wider population, i.e. routinely conducted testing and monitoring activities to identify and isolate infected individuals regardless of their clinical symptoms. Such active surveillance strategies will enable more effective prevention of the spread of the virus as they can generate more precise population-based parameters during a pandemic. This essential information will be required in order to determine the best strategic and targeted short-term countermeasures to limit infection spread locally.

Trial Design: This trial implements a cluster-randomised, two-factorial controlled, prospective, interventional, single-blinded design with four study arms, each representing a different SARS-CoV-2 testing and surveillance strategy.

Participants: Eligible are individuals age 7 years or older living in Germany's Rhein-Neckar Region who consent to provide a saliva sample (all four arms) after completion of a brief questionnaire (two arms only). For the qualitative component, different samples of study participants and non-participants (i.e. eligible for study, but refuse to participate) will be identified for additional interviews. For these interviews, only individuals age 18 years or older are eligible.

Intervention And Comparator: Of the four surveillance strategies to be assessed and compared, Strategy A1 is considered the gold standard for prevalence estimation and used to determine bias in other arms. To determine the cost-effectiveness, each strategy is compared to status quo, defined as the currently practiced passive surveillance approach. Strategy A1: Individuals (one per household) receive information and study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the sample is tested for SARS-CoV-2 using Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP). Strategy A2: Individuals (one per household) receive information and study material by mail with instructions on how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. Strategy B1: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms and risk exposures. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions on how to produce a saliva sample and how to return the sample by mail. Once received by the laboratory, the saliva sample is tested for SARS-CoV-2 using RT-LAMP. Strategy B2: Individuals (one per household) receive information by mail on how to complete a brief pre-screening questionnaire which asks about COVID-19 related clinical symptoms. Only individuals whose pre-screening score crosses a defined threshold, will then receive additional study material by mail with instructions how to produce their own as well as saliva samples from each household member and how to return these samples by mail. Once received by the laboratory, the samples are tested for SARS-CoV-2 using RT-LAMP. In each strategy, RT-LAMP positive samples are additionally analyzed with qPCR in order to minimize the number of false positives.

Main Outcomes: The identification of the one best strategy will be determined by a set of parameters. Primary outcomes include costs per correctly screened person, costs per positive case, positive detection rate, and precision of positive detection rate. Secondary outcomes include participation rate, costs per asymptomatic case, prevalence estimates, number of asymptomatic cases per study arm, ratio of symptomatic to asymptomatic cases per study arm, participant satisfaction. Additional study components (not part of the trial) include cost effectiveness of each of the four surveillance strategies compared to passive monitoring (i.e. status quo), development of a prognostic model to predict hospital utilization caused by SARS-CoV-2, time from test shipment to test application and time from test shipment to test result, and perception and preferences of the persons to be tested with regard to test strategies.

Randomisation: Samples are drawn in three batches of three continuous weeks. Randomisation follows a two-stage process. First, a total of 220 sampling points have been allocated to the three different batches. To obtain an integer solution, the Cox-algorithm for controlled rounding has been used. Afterwards, sample points have been drawn separately per batch, following a probability proportional to size (PPS) random sample. Second, for each cluster the same number of residential addresses is randomly sampled from the municipal registries (self-weighted sample of individuals). The 28,125 addresses drawn per municipality are then randomly allocated to the four study arms A1, A2, B1, and B2 in the ratio 5 to 2.5 to 14 to 7 based on the expected response rates in each arm and the sensitivity and specificity of the pre-screening tool as applied in strategy B1 and B2. Based on the assumptions, this allocation should yield 2500 saliva samples in each strategy. Although a municipality can be sampled by multiple batches and the overall number of addresses per municipality might vary, the number of addresses contacted in each arm is kept constant.

Blinding (masking): The design is single-blinded, meaning the staff conducting the SARS-CoV-2 tests are unaware of the study arm assignment of each single participant and test sample.

Sample Sizes: Total sample size for the trial is 10,000 saliva samples equally allocated to the four study arms (i.e. 2,500 participants per arm). For the qualitative component, up to 60 in-depth interviews will be conducted with about 30 study participants (up to 15 in each arm A and B) and 30 participation refusers (up to 15 in each arm A and B) purposefully selected from the quantitative study sample to represent a variety of gender and ages to explore experiences with admission or rejection of study participation. Up to 25 asymptomatic SARS-CoV-2 positive study participants will be purposefully selected to explore the way in which asymptomatic men and women diagnosed with SARS-CoV-2 give meaning to their diagnosis and to the dialectic between feeling concurrently healthy and yet also being at risk for transmitting COVID-19. In addition, 100 randomly selected study participants will be included to explore participants' perspective on testing processes and implementation.

Trial Status: Final protocol version is "Surveillance_Studienprotokoll_03Nov2020_v1_2" from November 3, 2020. Recruitment started November 18, 2020 and is expected to end by or before December 31, 2020.

Trial Registration: The trial is currently being registered with the German Clinical Trials Register (Deutsches Register Klinischer Studien), DRKS00023271 ( https://www.drks.de/drks_web/navigate.do?navigationId=trial . HTML&TRIAL_ID=DRKS00023271). Retrospectively registered 30 November 2020.

Full Protocol: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.
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http://dx.doi.org/10.1186/s13063-020-04982-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7791150PMC
January 2021

CRISPR/Cas12a-mediated labeling of MET receptor enables quantitative single-molecule imaging of endogenous protein organization and dynamics.

iScience 2021 Jan 7;24(1):101895. Epub 2020 Dec 7.

Single Molecule Biophysics, Institute of Physical and Theoretical Chemistry, Goethe University Frankfurt, Max-von-Laue Str. 7, 60438 Frankfurt, Germany.

Single-molecule localization microscopy (SMLM) reports on protein organization in cells with near-molecular resolution and in combination with stoichiometric labeling enables protein counting. Fluorescent proteins allow stoichiometric labeling of cellular proteins; however, most methods either lead to overexpression or are complex and time demanding. We introduce CRISPR/Cas12a for simple and efficient tagging of endogenous proteins with a photoactivatable protein for quantitative SMLM and single-particle tracking. We constructed a HEK293T cell line with the receptor tyrosine kinase MET tagged with mEos4b and demonstrate full functionality. We determine the oligomeric state of MET with quantitative SMLM and find a reorganization from monomeric to dimeric MET upon ligand stimulation. In addition, we measured the mobility of single MET receptors in resting and ligand-treated cells. The combination of CRISPR/Cas12a-assisted endogenous protein labeling and super-resolution microscopy represents a powerful tool for cell biological research with molecular resolution.
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http://dx.doi.org/10.1016/j.isci.2020.101895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753144PMC
January 2021

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Viruses 2020 08 7;12(8). Epub 2020 Aug 7.

Center of Infectious Diseases, Virology, Heidelberg University Hospital, 69120 Heidelberg, Germany.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.
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http://dx.doi.org/10.3390/v12080863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472728PMC
August 2020

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.

Sci Transl Med 2020 08 27;12(556). Epub 2020 Jul 27.

Center for Molecular Biology of Heidelberg University (ZMBH), Heidelberg, Germany.

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.
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http://dx.doi.org/10.1126/scitranslmed.abc7075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574920PMC
August 2020

CRISPR-Cas12a-assisted PCR tagging of mammalian genes.

J Cell Biol 2020 06;219(6)

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), German Cancer Research Center (DKFZ)-ZMBH Alliance, Heidelberg, Germany.

Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.
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http://dx.doi.org/10.1083/jcb.201910210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7265327PMC
June 2020

Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast.

Nat Commun 2019 07 4;10(1):2960. Epub 2019 Jul 4.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, 69120, Heidelberg, Germany.

Clone collections of modified strains ("libraries") are a major resource for systematic studies with the yeast Saccharomyces cerevisiae. Construction of such libraries is time-consuming, costly and confined to the genetic background of a specific yeast strain. To overcome these limitations, we present CRISPR-Cas12a (Cpf1)-assisted tag library engineering (CASTLING) for multiplexed strain construction. CASTLING uses microarray-synthesized oligonucleotide pools and in vitro recombineering to program the genomic insertion of long DNA constructs via homologous recombination. One simple transformation yields pooled libraries with >90% of correctly tagged clones. Up to several hundred genes can be tagged in a single step and, on a genomic scale, approximately half of all genes are tagged with only ~10-fold oversampling. We report several parameters that affect tagging success and provide a quantitative targeted next-generation sequencing method to analyze such pooled collections. Thus, CASTLING unlocks avenues for increasing throughput in functional genomics and cell biology research.
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http://dx.doi.org/10.1038/s41467-019-10816-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609715PMC
July 2019

YeastRGB: comparing the abundance and localization of yeast proteins across cells and libraries.

Nucleic Acids Res 2019 01;47(D1):D1245-D1249

Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel.

The ability to measure the abundance and visualize the localization of proteins across the yeast proteome has stimulated hypotheses on gene function and fueled discoveries. While the classic C' tagged GFP yeast library has been the only resource for over a decade, the recent development of the SWAT technology has led to the creation of multiple novel yeast libraries where new-generation fluorescent reporters are fused at the N' and C' of open reading frames. Efficient access to these data requires a user interface to visualize and compare protein abundance, localization and co-localization across cells, strains, and libraries. YeastRGB (www.yeastRGB.org) was designed to address such a need, through a user-friendly interface that maximizes informative content. It employs a compact display where cells are cropped and tiled together into a 'cell-grid.' This representation enables viewing dozens of cells for a particular strain within a display unit, and up to 30 display units can be arrayed on a standard high-definition screen. Additionally, the display unit allows users to control zoom-level and overlay of images acquired using different color channels. Thus, YeastRGB makes comparing abundance and localization efficient, across thousands of cells from different strains and libraries.
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http://dx.doi.org/10.1093/nar/gky941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6324022PMC
January 2019

Monitoring Protein Dynamics in Protein -Mannosyltransferase Mutants In Vivo by Tandem Fluorescent Protein Timers.

Molecules 2018 Oct 12;23(10). Epub 2018 Oct 12.

Centre for Organismal Studies (COS), Heidelberg University, 69120 Heidelberg, Germany.

For proteins entering the secretory pathway, a major factor contributing to maturation and homeostasis is glycosylation. One relevant type of protein glycosylation is -mannosylation, which is essential and evolutionarily-conserved in fungi, animals, and humans. Our recent proteome-wide study in the eukaryotic model organism revealed that more than 26% of all proteins entering the secretory pathway receive -mannosyl glycans. In a first attempt to understand the impact of -mannosylation on these proteins, we took advantage of a tandem fluorescent timer (tFT) reporter to monitor different aspects of protein dynamics. We analyzed tFT-reporter fusions of 137 unique -mannosylated proteins, mainly of the secretory pathway and the plasma membrane, in mutants lacking the major protein -mannosyltransferases Pmt1, Pmt2, or Pmt4. In these three Δ mutants, a total of 39 individual proteins were clearly affected, and Pmt-specific substrate proteins could be identified. We observed that -mannosylation may cause both enhanced and diminished protein abundance and/or stability when compromised, and verified our findings on the examples of Axl2-tFT and Kre6-tFT fusion proteins. The identified target proteins are a valuable resource towards unraveling the multiple functions of -mannosylation at the molecular level.
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http://dx.doi.org/10.3390/molecules23102622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222916PMC
October 2018

Genome-wide C-SWAT library for high-throughput yeast genome tagging.

Nat Methods 2018 08 9;15(8):598-600. Epub 2018 Jul 9.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.

Here we describe a C-SWAT library for high-throughput tagging of Saccharomyces cerevisiae open reading frames (ORFs). In 5,661 strains, we inserted an acceptor module after each ORF that can be efficiently replaced with tags or regulatory elements. We validated the library with targeted sequencing and tagged the proteome with bright fluorescent proteins to quantify the effect of heterologous transcription terminators on protein expression and to localize previously undetected proteins.
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http://dx.doi.org/10.1038/s41592-018-0045-8DOI Listing
August 2018

Upregulation of SPS100 gene expression by an antisense RNA via a switch of mRNA isoforms with different stabilities.

Nucleic Acids Res 2017 Nov;45(19):11144-11158

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany.

Pervasive transcription of genomes generates multiple classes of non-coding RNAs. One of these classes are stable long non-coding RNAs which overlap coding genes in antisense direction (asRNAs). The function of such asRNAs is not fully understood but several cases of antisense-dependent gene expression regulation affecting the overlapping genes have been demonstrated. Using high-throughput yeast genetics and a limited set of four growth conditions we previously reported a regulatory function for ∼25% of asRNAs, most of which repress the expression of the sense gene. To further explore the roles of asRNAs we tested more conditions and identified 15 conditionally antisense-regulated genes, 6 of which exhibited antisense-dependent enhancement of gene expression. We focused on the sporulation-specific gene SPS100, which becomes upregulated upon entry into starvation or sporulation as a function of the antisense transcript SUT169. We demonstrate that the antisense effect is mediated by its 3' intergenic region (3'-IGR) and that this regulation can be transferred to other genes. Genetic analysis revealed that SUT169 functions by changing the relative expression of SPS100 mRNA isoforms from a short and unstable transcript to a long and stable species. These results suggest a novel mechanism of antisense-dependent gene regulation via mRNA isoform switching.
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http://dx.doi.org/10.1093/nar/gkx737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737743PMC
November 2017

Alterations in cellular metabolism triggered by or inactivation cause imbalanced dNTP pools and increased mutagenesis.

Proc Natl Acad Sci U S A 2017 05 17;114(22):E4442-E4451. Epub 2017 Apr 17.

German Cancer Research Center, 69120 Heidelberg, Germany;

Eukaryotic DNA replication fidelity relies on the concerted action of DNA polymerase nucleotide selectivity, proofreading activity, and DNA mismatch repair (MMR). Nucleotide selectivity and proofreading are affected by the balance and concentration of deoxyribonucleotide (dNTP) pools, which are strictly regulated by ribonucleotide reductase (RNR). Mutations preventing DNA polymerase proofreading activity or MMR function cause mutator phenotypes and consequently increased cancer susceptibility. To identify genes not previously linked to high-fidelity DNA replication, we conducted a genome-wide screen in using DNA polymerase active-site mutants as a "sensitized mutator background." Among the genes identified in our screen, three metabolism-related genes (, , and ) have not been previously associated to the suppression of mutations. Loss of either the transcription factor Gln3 or inactivation of the CTP synthetase Ura7 both resulted in the activation of the DNA damage response and imbalanced dNTP pools. Importantly, these dNTP imbalances are strongly mutagenic in genetic backgrounds where DNA polymerase function or MMR activity is partially compromised. Previous reports have shown that dNTP pool imbalances can be caused by mutations altering the allosteric regulation of enzymes involved in dNTP biosynthesis (e.g., RNR or dCMP deaminase). Here, we provide evidence that mutations affecting genes involved in RNR substrate production can cause dNTP imbalances, which cannot be compensated by RNR or other enzymatic activities. Moreover, Gln3 inactivation links nutrient deprivation to increased mutagenesis. Our results suggest that similar genetic interactions could drive mutator phenotypes in cancer cells.
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http://dx.doi.org/10.1073/pnas.1618714114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465912PMC
May 2017

The regulatable MAL32 promoter in Saccharomyces cerevisiae: characteristics and tools to facilitate its use.

Yeast 2017 01 23;34(1):39-49. Epub 2016 Nov 23.

Zentrum für Molekulare Biologie der Universität Heidelberg, University of Heidelberg, Im Neuenheimer Feld 282, 69120, Heidelberg, Germany.

Here we describe a set of tools to facilitate the use of maltose and the MAL32 promoter for regulated gene expression in yeast, alone or in combination with the GAL1 promoter. Using fluorescent protein reporters we find that under non-inducing conditions the MAL32 promoter exhibits a low basal level of expression, similar to the GAL1 promoter, and that both promoters can be induced independently of each other using the respective sugars, maltose and galactose. While their repression upon glucose addition is immediate and complete, we found that the MAL32 and GAL1 promoters each exhibit distinct induction kinetics. A set of plasmids is available to facilitate the application of the MAL32 promoter for chromosomal modifications using PCR targetting and for plasmid based gene expression. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/yea.3214DOI Listing
January 2017

Protein Abundance Control by Non-coding Antisense Transcription.

Cell Rep 2016 06 9;15(12):2625-36. Epub 2016 Jun 9.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany; Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. Electronic address:

Stable unannotated transcripts (SUTs), some of which overlap protein-coding genes in antisense direction, are a class of non-coding RNAs. While case studies have reported important regulatory roles for several of such RNAs, their general impact on protein abundance regulation of the overlapping gene is not known. To test this, we employed seamless gene manipulation to repress antisense SUTs of 162 yeast genes by using a unidirectional transcriptional terminator and a GFP tag. We found that the mere presence of antisense SUTs was not sufficient to influence protein abundance, that observed effects of antisense SUTs correlated with sense transcript start site overlap, and that the effects were generally weak and led to reduced protein levels. Antisense regulated genes showed increased H3K4 di- and trimethylation and had slightly lower than expected noise levels. Our results suggest that the functionality of antisense RNAs has gene and condition-specific components.
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http://dx.doi.org/10.1016/j.celrep.2016.05.043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920891PMC
June 2016

One library to make them all: streamlining the creation of yeast libraries via a SWAp-Tag strategy.

Nat Methods 2016 Apr 29;13(4):371-378. Epub 2016 Feb 29.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist, as their construction is extremely expensive and laborious. To overcome these limitations, we developed a SWAp-Tag (SWAT) method that enables one parental library to be modified easily and efficiently to give rise to an endless variety of libraries of choice. To showcase the versatility of the SWAT approach, we constructed and investigated a library of ∼1,800 strains carrying SWAT-GFP modules at the amino termini of endomembrane proteins and then used it to create two new libraries (mCherry and seamless GFP). Our work demonstrates how the SWAT method allows fast and effortless creation of yeast libraries, opening the door to new ways of systematically studying cell biology.
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http://dx.doi.org/10.1038/nmeth.3795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4869835PMC
April 2016

Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.

Mol Biol Cell 2016 Jan 25;27(2):360-70. Epub 2015 Nov 25.

Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany

Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.
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http://dx.doi.org/10.1091/mbc.E15-07-0525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4713137PMC
January 2016

Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein.

Mol Biol Cell 2015 Jun 15;26(11):2054-66. Epub 2015 Apr 15.

Department of Systems Biology, Harvard Medical School, Boston, MA 02115 Renal Division, Brigham and Women's Hospital, Boston, MA 02115

We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (∼180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (∼120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein-protein interactions. We also use MPE-FCCS to detect drug-protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1/hmKeima8.5 and C343/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.
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http://dx.doi.org/10.1091/mbc.E14-10-1473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472016PMC
June 2015

Protein quality control at the inner nuclear membrane.

Nature 2014 Dec;516(7531):410-3

1] Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany [2] Cell Morphogenesis and Signal Transduction, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.
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http://dx.doi.org/10.1038/nature14096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493439PMC
December 2014

PCR Duplication: A One-Step Cloning-Free Method to Generate Duplicated Chromosomal Loci and Interference-Free Expression Reporters in Yeast.

PLoS One 2014 10;9(12):e114590. Epub 2014 Dec 10.

Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Allianz, University of Heidelberg, 69120, Heidelberg, Germany.

Here, we report on a novel PCR targeting-based strategy called 'PCR duplication' that enables targeted duplications of genomic regions in the yeast genome using a simple PCR-based approach. To demonstrate its application we first duplicated the promoter of the FAR1 gene in yeast and simultaneously inserted a GFP downstream of it. This created a reporter for promoter activity while leaving the FAR1 gene fully intact. In another experiment, we used PCR duplication to increase the dosage of a gene in a discrete manner, from 1× to 2x. Using TUB4, the gene encoding for the yeast γ-tubulin, we validated that this led to corresponding increases in the levels of mRNA and protein. PCR duplication is an easy one-step procedure that can be adapted in different ways to permit rapid, disturbance-free investigation of various genomic regulatory elements without the need for ex vivo cloning.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114590PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262419PMC
October 2017

Quantification of cytosolic interactions identifies Ede1 oligomers as key organizers of endocytosis.

Mol Syst Biol 2014 Nov 3;10:756. Epub 2014 Nov 3.

European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Clathrin-mediated endocytosis is a highly conserved intracellular trafficking pathway that depends on dynamic protein-protein interactions between up to 60 different proteins. However, little is known about the spatio-temporal regulation of these interactions. Using fluorescence (cross)-correlation spectroscopy in yeast, we tested 41 previously reported interactions in vivo and found 16 to exist in the cytoplasm. These detected cytoplasmic interactions included the self-interaction of Ede1, homolog of mammalian Eps15. Ede1 is the crucial scaffold for the organization of the early stages of endocytosis. We show that oligomerization of Ede1 through its central coiled coil domain is necessary for its localization to the endocytic site and we link the oligomerization of Ede1 to its function in locally concentrating endocytic adaptors and organizing the endocytic machinery. Our study sheds light on the importance of the regulation of protein-protein interactions in the cytoplasm for the assembly of the endocytic machinery in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4299599PMC
http://dx.doi.org/10.15252/msb.20145422DOI Listing
November 2014

Tandem fluorescent protein timers for in vivo analysis of protein dynamics.

Nat Biotechnol 2012 Jun 24;30(7):708-14. Epub 2012 Jun 24.

Center for Molecular Biology of the University of Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.

The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.
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http://dx.doi.org/10.1038/nbt.2281DOI Listing
June 2012

Seamless gene tagging by endonuclease-driven homologous recombination.

PLoS One 2011 22;6(8):e23794. Epub 2011 Aug 22.

Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.

Gene tagging facilitates systematic genomic and proteomic analyses but chromosomal tagging typically disrupts gene regulatory sequences. Here we describe a seamless gene tagging approach that preserves endogenous gene regulation and is potentially applicable in any species with efficient DNA double-strand break repair by homologous recombination. We implement seamless tagging in Saccharomyces cerevisiae and demonstrate its application for protein tagging while preserving simultaneously upstream and downstream gene regulatory elements. Seamless tagging is compatible with high-throughput strain construction using synthetic genetic arrays (SGA), enables functional analysis of transcription antisense to open reading frames and should facilitate systematic and minimally-invasive analysis of gene functions.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0023794PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3161820PMC
April 2012

Artificial tethering to nuclear pores promotes partitioning of extrachromosomal DNA during yeast asymmetric cell division.

Curr Biol 2011 Jan;21(1):R17-8

Summary: Asymmetric cell division in unicellular organisms enables sequestration of senescence factors to specific subpopulations. Accumulation of autonomously replicating sequence (ARS) plasmids, which frequently emerge from recombination within the highly repetitive ribosomal DNA locus, is linked to limited replicative life span of Saccharomyces cerevisiae cells [1]. During budding yeast cell division, ARS plasmids are retained in the ageing mother cell, such that only 1 out of 10 plasmids enters the rejuvenated bud [2]. Binding of ARS plasmids to nuclear structures retained in the mother cell was speculated to explain asymmetric plasmid segregation [2]. Association with nuclear pore complexes (NPCs) was proposed to underlie retention of ARS plasmids in the mother cell [3]. However, the role of NPCs in segregation of ARS plasmids is unclear, as NPCs are partitioned between mother and bud nuclei during mitosis [4,5]. Here we analyzed how segregation of ARS plasmids is influenced by their interaction with NPCs. We found that artificial tethering to NPCs promotes transport of ARS plasmids into the bud. Moreover, our experiments provide support for the notion that interaction with ARS plasmids does not affect movement of NPCs into the bud. We conclude that binding to NPCs cannot by itself contribute to asymmetric segregation of ARS plasmids.
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http://dx.doi.org/10.1016/j.cub.2010.11.034DOI Listing
January 2011
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