Publications by authors named "Stefanie Kellner"

48 Publications

Insomnia Associated with Tinnitus and Gender Differences.

Int J Environ Res Public Health 2021 03 19;18(6). Epub 2021 Mar 19.

KSM Clinic for Sleep Medicine Bad Zurzach, 5330 Bad Zurzach, Switzerland.

Chronic tinnitus causes a decrease in well-being and can negatively affect sleep quality. It has further been indicated that there are clinically relevant gender differences, which may also have an impact on sleep quality. By conducting a retrospective and explorative data analysis for differences in patients with tinnitus and patients diagnosed with tinnitus and insomnia, hypothesized differences were explored in the summed test scores and on item-level of the validated psychometric instruments. A cross-sectional study was conducted collecting data from a sample of tinnitus patients ( = 76). Insomnia was diagnosed in 49 patients. Gender differences were found on aggregated test scores of the MADRS and BDI with men scoring higher than women, indicating higher depressive symptoms in men. Women stated to suffer more from headaches ( < 0.003), neck pain ( < 0.006) and nervousness as well as restlessness ( < 0.02). Women also reported an increase in tinnitus loudness in response to stress compared to men ( < 0.03). Male individuals with tinnitus and insomnia have higher depression scores and more clinically relevant depressive symptoms than women, who suffer more from psychosomatic symptoms. The results indicate a need for a targeted therapy of depressive symptoms in male patients and targeted treatment of psychosomatic symptoms, stress-related worsening of insomnia and tinnitus in women.
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http://dx.doi.org/10.3390/ijerph18063209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8003647PMC
March 2021

mU54 tRNA Hypomodification by Lack of TRMT2A Drives the Generation of tRNA-Derived Small RNAs.

Int J Mol Sci 2021 Mar 14;22(6). Epub 2021 Mar 14.

Institute of Biomedicine (iBiMED), Department of Medical Sciences, University of Aveiro, 3810 Aveiro, Portugal.

Transfer RNA (tRNA) molecules contain various post-transcriptional modifications that are crucial for tRNA stability, translation efficiency, and fidelity. Besides their canonical roles in translation, tRNAs also originate tRNA-derived small RNAs (tsRNAs), a class of small non-coding RNAs with regulatory functions ranging from translation regulation to gene expression control and cellular stress response. Recent evidence indicates that tsRNAs are also modified, however, the impact of tRNA epitranscriptome deregulation on tsRNAs generation is only now beginning to be uncovered. The 5-methyluridine (mU) modification at position 54 of cytosolic tRNAs is one of the most common and conserved tRNA modifications among species. The tRNA methyltransferase TRMT2A catalyzes this modification, but its biological role remains mostly unexplored. Here, we show that TRMT2A knockdown in human cells induces mU54 tRNA hypomodification and tsRNA formation. More specifically, mU54 hypomodification is followed by overexpression of the ribonuclease angiogenin (ANG) that cleaves tRNAs near the anticodon, resulting in accumulation of 5'tRNA-derived stress-induced RNAs (5'tiRNAs), namely 5'tiRNA-Gly and 5'tiRNA-Glu, among others. Additionally, transcriptomic analysis confirms that down-regulation of TRMT2A and consequently mU54 hypomodification impacts the cellular stress response and RNA stability, which is often correlated with tiRNA generation. Accordingly, exposure to oxidative stress conditions induces TRMT2A down-regulation and tiRNA formation in mammalian cells. These results establish a link between tRNA hypomethylation and ANG-dependent tsRNAs formation and unravel mU54 as a tRNA cleavage protective mark.
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http://dx.doi.org/10.3390/ijms22062941DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8001983PMC
March 2021

Instrumental analysis of RNA modifications.

Crit Rev Biochem Mol Biol 2021 Apr 22;56(2):178-204. Epub 2021 Feb 22.

Department of Chemistry, Ludwig Maximilians University, Munich, Germany.

Organisms from all domains of life invest a substantial amount of energy for the introduction of RNA modifications into nearly all transcripts studied to date. Instrumental analysis of RNA can focus on the modified residues and reveal the function of these epitranscriptomic marks. Here, we will review recent advances and breakthroughs achieved by NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome.
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http://dx.doi.org/10.1080/10409238.2021.1887807DOI Listing
April 2021

Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo.

Nat Commun 2021 01 15;12(1):389. Epub 2021 Jan 15.

Department of Chemistry, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, 81377, Munich, Germany.

Recently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNA and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.
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http://dx.doi.org/10.1038/s41467-020-20576-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7810713PMC
January 2021

Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6.

Nucleic Acids Res 2021 01;49(2):1006-1022

German Cancer Research Center - Deutsches Krebsforschungszentrum (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.
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http://dx.doi.org/10.1093/nar/gkaa1193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826283PMC
January 2021

Chemical Amination/Imination of Carbonothiolated Nucleosides During RNA Hydrolysis.

Angew Chem Int Ed Engl 2021 02 10;60(8):3961-3966. Epub 2020 Dec 10.

Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH, 45221-0172, USA.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold-standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state-of-the-art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non-natural mcm isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher-energy collisional dissociation (HCD)-based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium-buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications.
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http://dx.doi.org/10.1002/anie.202010793DOI Listing
February 2021

METTL6 is a tRNA mC methyltransferase that regulates pluripotency and tumor cell growth.

Sci Adv 2020 Aug 26;6(35):eaaz4551. Epub 2020 Aug 26.

Institute of Functional Epigenetics, Helmholtz Zentrum München, 85764 Neuherberg, Germany.

Recently, covalent modifications of RNA, such as methylation, have emerged as key regulators of all aspects of RNA biology and have been implicated in numerous diseases, for instance, cancer. Here, we undertook a combination of in vitro and in vivo screens to test 78 potential methyltransferases for their roles in hepatocellular carcinoma (HCC) cell proliferation. We identified methyltransferase-like protein 6 (METTL6) as a crucial regulator of tumor cell growth. We show that METTL6 is a bona fide transfer RNA (tRNA) methyltransferase, catalyzing the formation of 3-methylcytidine at C32 of specific serine tRNA isoacceptors. Deletion of in mouse stem cells results in changes in ribosome occupancy and RNA levels, as well as impaired pluripotency. In mice, knockout results in reduced energy expenditure. We reveal a previously unknown pathway in the maintenance of translation efficiency with a role in maintaining stem cell self-renewal, as well as impacting tumor cell growth profoundly.
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http://dx.doi.org/10.1126/sciadv.aaz4551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449687PMC
August 2020

tRNA-modifying enzyme mutations induce codon-specific mistranslation and protein aggregation in yeast.

RNA Biol 2021 04 17;18(4):563-575. Epub 2020 Sep 17.

Department of Medical Sciences, Institute of Biomedicine - iBiMED, University of Aveiro, Aveiro, Portugal.

Protein synthesis rate and accuracy are tightly controlled by the cell and are essential for proteome homoeostasis (proteostasis); however, the full picture of how mRNA translational factors maintain protein synthesis accuracy and co-translational protein folding are far from being fully understood. To address this question, we evaluated the role of 70 yeast tRNA-modifying enzyme genes on protein aggregation and used mass spectrometry to identify the aggregated proteins. We show that modification of uridine at anticodon position 34 (U34) by the tRNA-modifying enzymes Elp1, Elp3, Sml3 and Trm9 is critical for proteostasis, the mitochondrial tRNA-modifying enzyme Slm3 plays a fundamental role in general proteostasis and that stress response proteins whose genes are enriched in codons decoded by tRNAs lacking mcmU, mcmsU, ncmU, ncmUm, modifications are overrepresented in protein aggregates of the and KO strains. Increased rates of amino acid misincorporation were also detected in these strains at protein sites that specifically mapped to the codons sites that are decoded by the hypomodified tRNAs, demonstrating that U tRNA modifications safeguard the proteome from translational errors, protein misfolding and proteotoxic stress.
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http://dx.doi.org/10.1080/15476286.2020.1819671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7971265PMC
April 2021

Identification and rescue of a tRNA wobble inosine deficiency causing intellectual disability disorder.

RNA 2020 11 6;26(11):1654-1666. Epub 2020 Aug 6.

Department of Biology, Center for RNA Biology, University of Rochester, Rochester, New York 14627, USA.

The deamination of adenosine to inosine at the wobble position of tRNA is an essential post-transcriptional RNA modification required for wobble decoding in bacteria and eukaryotes. In humans, the wobble inosine modification is catalyzed by the heterodimeric ADAT2/3 complex. Here, we describe novel pathogenic ADAT3 variants impairing adenosine deaminase activity through a distinct mechanism that can be corrected through expression of the heterodimeric ADAT2 subunit. The variants were identified in a family in which all three siblings exhibit intellectual disability linked to biallelic variants in the locus. The biallelic ADAT3 variants result in a missense variant converting alanine to valine at a conserved residue or the introduction of a premature stop codon in the deaminase domain. Fibroblast cells derived from two ID-affected individuals exhibit a reduction in tRNA wobble inosine levels and severely diminished adenosine tRNA deaminase activity. Notably, the ADAT3 variants exhibit impaired interaction with the ADAT2 subunit and alterations in ADAT2-dependent nuclear localization. Based upon these findings, we find that tRNA adenosine deaminase activity and wobble inosine modification can be rescued in patient cells by overexpression of the ADAT2 catalytic subunit. These results uncover a key role for the inactive ADAT3 deaminase domain in proper assembly with ADAT2 and demonstrate that ADAT2/3 nuclear import is required for maintaining proper levels of the wobble inosine modification in tRNA.
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http://dx.doi.org/10.1261/rna.076380.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566568PMC
November 2020

Eukaryotic life without tQCUG: the role of Elongator-dependent tRNA modifications in Dictyostelium discoideum.

Nucleic Acids Res 2020 08;48(14):7899-7913

Ribogenetics Biochemistry Lab, Department of Life Sciences and Chemistry, Jacobs University Bremen gGmbH, DE 28759 Bremen, Germany.

In the Elongator-dependent modification pathway, chemical modifications are introduced at the wobble uridines at position 34 in transfer RNAs (tRNAs), which serve to optimize codon translation rates. Here, we show that this three-step modification pathway exists in Dictyostelium discoideum, model of the evolutionary superfamily Amoebozoa. Not only are previously established modifications observable by mass spectrometry in strains with the most conserved genes of each step deleted, but also additional modifications are detected, indicating a certain plasticity of the pathway in the amoeba. Unlike described for yeast, D. discoideum allows for an unconditional deletion of the single tQCUG gene, as long as the Elongator-dependent modification pathway is intact. In gene deletion strains of the modification pathway, protein amounts are significantly reduced as shown by flow cytometry and Western blotting, using strains expressing different glutamine leader constructs fused to GFP. Most dramatic are these effects, when the tQCUG gene is deleted, or Elp3, the catalytic component of the Elongator complex is missing. In addition, Elp3 is the most strongly conserved protein of the modification pathway, as our phylogenetic analysis reveals. The implications of this observation are discussed with respect to the evolutionary age of the components acting in the Elongator-dependent modification pathway.
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http://dx.doi.org/10.1093/nar/gkaa560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430636PMC
August 2020

Sleep disorders in migrants and refugees: a systematic review with implications for personalized medical approach.

EPMA J 2020 Jun 13;11(2):251-260. Epub 2020 May 13.

Institute of Cognitive Science, Woosuk University, Wanju, South Korea.

Background: Sleep disorders are very common in migrants and refugees, often as a comorbid disorder to different somatic or psychiatric diagnoses and psychological disturbances such as metabolic syndrome, post-traumatic stress disorder, depression, and anxiety disorders.

Objectives: To review published prevalence rates as well as possible predictors for sleep disturbances in these vulnerable groups, including pre-migration stress, acculturation, and trauma before, during, and after migration, integration, and lifestyle in the host country with implications for predictive, preventive, and personalized medical approach (3PM).

Data Sources: Electronic databases PubMed, PsycInfo, and Web of Knowledge were searched using (combined) search terms "migrant," "asylum seeker," "refugee," "sleep disturbances," "sleep disorder," "insomnia," and "sleep wake disorder."

Study Eligibility Criteria: Peer-reviewed studies from 2000 to 2018 reporting data on prevalence and/or predictors of any measure of sleep disturbance were included.

Participants: Studies on international migrants and refugees, as well as internally displaced populations, were included.

Methods: We conducted a systematic review on the topic of sleep disorders in migrant and refugee populations. Only published articles and reviews in peer-reviewed journals were included.

Results: We analyzed five studies on sleep disorders in migrants, five studies on adult refugees, and three on refugee children and adolescents. Prevalence of sleep disorders in migrants and refugees ranges between 39 and 99%. In migrant workers, stress related to integration and adaptation to the host society is connected to higher risks of snoring, metabolic diseases, and insomnia. Sleep disturbances in refugees are predicted by past war experience. Sleep difficulties in adult and child refugees are strongly correlated to trauma. Torture of parents and grandparents can predict sleep disorders in refugee children, while being accompanied by parents to the host country has a protective effect on children's sleep.

Conclusions And Implications: Considering the differences in risk factors, vulnerability, and traumatic life events for different migrant populations, origins of sleep difficulties vary, depending on the migrant populations. Effects on sleep disturbances and sleep quality may be a result of integration in the host country, including changes of lifestyle, such as diet and working hours with implication for OSAS (obstructive sleep apnea) and insomnia. Compared with migrant populations, sleep disturbances in refugee populations are more correlated with mental health symptoms and disorders, especially PTSD (post-traumatic stress disorder), than with psychosocial problems. In juvenile refugee populations, psychological problems and disturbed sleep are associated with traumatic experiences during their journey to the host country. Findings highlight the need for expert recommendations for development of 3P approach stratified in the following: (1) prediction, including structured exploration of predisposing and precipitating factors that may trigger acute insomnia, screening of the according sleep disorders by validated translated questionnaires and sleep diaries, and a face-to-face or virtual setting and screening of OSAS; (2) target prevention by sleep health education for female and male refugees and migrant workers, including shift workers; and (3) personalized medical approach, including translated cognitive behavioral treatment for insomnia (CBT-I) and imagery rehearsal therapy for refugees and telehealth programs for improved CPAP adherence in migrants, with the goal to enable better sleep health quality and improved health economy.
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http://dx.doi.org/10.1007/s13167-020-00205-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272531PMC
June 2020

Synthesis and Metabolic Fate of 4-Methylthiouridine in Bacterial tRNA.

Chembiochem 2020 10 18;21(19):2768-2771. Epub 2020 Jun 18.

Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.

Ribonucleic acid (RNA) is central to many life processes and, to fulfill its function, it has a substantial chemical variety in its building blocks. Enzymatic thiolation of uridine introduces 4-thiouridine (s U) into many bacterial transfer RNAs (tRNAs), which is used as a sensor for UV radiation. A similar modified nucleoside, 2-thiocytidine, was recently found to be sulfur-methylated especially in bacteria exposed to antibiotics and simple methylating reagents. Herein, we report the synthesis of 4-methylthiouridine (ms U) and confirm its presence and additional formation under stress in Escherichia coli. We used the synthetic ms U for isotope dilution mass spectrometry and compared its abundance to other reported tRNA damage products. In addition, we applied sophisticated stable-isotope pulse chase studies (NAIL-MS) and showed its AlkB-independent removal in vivo. Our findings reveal the complex nature of bacterial RNA damage repair.
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http://dx.doi.org/10.1002/cbic.202000272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7586944PMC
October 2020

The rRNA mA methyltransferase METTL5 is involved in pluripotency and developmental programs.

Genes Dev 2020 05 26;34(9-10):715-729. Epub 2020 Mar 26.

Department of Biology II, Human Biology, and BioImaging, Ludwig-Maximilians Universität München, Munich 81377, Germany.

Covalent chemical modifications of cellular RNAs directly impact all biological processes. However, our mechanistic understanding of the enzymes catalyzing these modifications, their substrates and biological functions, remains vague. Amongst RNA modifications N-methyladenosine (mA) is widespread and found in messenger (mRNA), ribosomal (rRNA), and noncoding RNAs. Here, we undertook a systematic screen to uncover new RNA methyltransferases. We demonstrate that the methyltransferase-like 5 (METTL5) protein catalyzes mA in rRNA at position A We report that absence of in mouse embryonic stem cells (mESCs) results in a decrease in global translation rate, spontaneous loss of pluripotency, and compromised differentiation potential. METTL5-deficient mice are born at non-Mendelian rates and develop morphological and behavioral abnormalities. Importantly, mice lacking METTL5 recapitulate symptoms of patients with DNA variants in , thereby providing a new mouse disease model. Overall, our biochemical, molecular, and in vivo characterization highlights the importance of mA in rRNA in stemness, differentiation, development, and diseases.
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http://dx.doi.org/10.1101/gad.333369.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7197354PMC
May 2020

Synthesis of Galactosyl-Queuosine and Distribution of Hypermodified Q-Nucleosides in Mouse Tissues.

Angew Chem Int Ed Engl 2020 07 21;59(30):12352-12356. Epub 2020 Apr 21.

Department of Chemistry, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany.

Queuosine (Q) is a hypermodified RNA nucleoside that is found in tRNA , tRNA , tRNA , and tRNA . It is located at the wobble position of the tRNA anticodon loop, where it can interact with U as well as C bases located at the respective position of the corresponding mRNA codons. In tRNA and tRNA of higher eukaryotes, including humans, the Q base is for yet unknown reasons further modified by the addition of a galactose and a mannose sugar, respectively. The reason for this additional modification, and how the sugar modification is orchestrated with Q formation and insertion, is unknown. Here, we report a total synthesis of the hypermodified nucleoside galactosyl-queuosine (galQ). The availability of the compound enabled us to study the absolute levels of the Q-family nucleosides in six different organs of newborn and adult mice, and also in human cytosolic tRNA. Our synthesis now paves the way to a more detailed analysis of the biological function of the Q-nucleoside family.
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http://dx.doi.org/10.1002/anie.202002295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7384130PMC
July 2020

Production and purification of endogenously modified tRNA-derived small RNAs.

RNA Biol 2020 08 5;17(8):1104-1115. Epub 2020 Mar 5.

Division of Cell and Developmental Biology, Center for Anatomy and Cell Biology, Medical University Vienna , Vienna, Austria.

During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions.
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http://dx.doi.org/10.1080/15476286.2020.1733798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549616PMC
August 2020

Broadly applicable oligonucleotide mass spectrometry for the analysis of RNA writers and erasers in vitro.

Nucleic Acids Res 2020 04;48(7):e41

Department of Chemistry, Ludwig Maximilians University Munich, Butenandtstrasse 5-13, 81377 Munich, Germany.

RNAs are post-transcriptionally modified by dedicated writer or eraser enzymes that add or remove specific modifications, respectively. Mass spectrometry (MS) of RNA is a useful tool to study the modification state of an oligonucleotide (ON) in a sensitive manner. Here, we developed an ion-pairing reagent free chromatography for positive ion detection of ONs by low- and high-resolution MS, which does not interfere with other types of small compound analyses done on the same instrument. We apply ON-MS to determine the ONs from an RNase T1 digest of in vitro transcribed tRNA, which are purified after ribozyme-fusion transcription by automated size exclusion chromatography. The thus produced tRNAValAAC is substrate of the human tRNA ADAT2/3 enzyme and we confirm the deamination of adenosine to inosine and the formation of tRNAValIACin vitro by ON-MS. Furthermore, low resolution ON-MS is used to monitor the demethylation of ONs containing 1-methyladenosine by bacterial AlkB in vitro. The power of high-resolution ON-MS is demonstrated by the detection and mapping of modified ONs from native total tRNA digested with RNase T1. Overall, we present an oligonucleotide MS method which is broadly applicable to monitor in vitro RNA (de-)modification processes and native RNA.
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http://dx.doi.org/10.1093/nar/gkaa091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144906PMC
April 2020

[Treatment of insomnia in old age].

Z Gerontol Geriatr 2020 Mar 21;53(2):105-111. Epub 2020 Jan 21.

Fakultät für Gesundheit, Universität Witten Herdecke, Witten, Deutschland.

Insomnia is one of the most frequent health disorders in old age. It causes suffering and numerous health problems. Therefore, treatment is often indicated. Behavioral therapy is the treatment of choice even in older individuals. In addition, light therapy also has an important role. Pharmacological treatment measures are less well studied, the benefits in long-term use are unclear and should only be applied in the short term to reduce suffering as well as being integrated into a comprehensive treatment concept.
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http://dx.doi.org/10.1007/s00391-019-01684-3DOI Listing
March 2020

Identification of the 3-amino-3-carboxypropyl (acp) transferase enzyme responsible for acp3U formation at position 47 in Escherichia coli tRNAs.

Nucleic Acids Res 2020 02;48(3):1435-1450

Institute for Molecular Biosciences, Goethe-Universität Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt/M., Germany.

tRNAs from all domains of life contain modified nucleotides. However, even for the experimentally most thoroughly characterized model organism Escherichia coli not all tRNA modification enzymes are known. In particular, no enzyme has been found yet for introducing the acp3U modification at position 47 in the variable loop of eight E. coli tRNAs. Here we identify the so far functionally uncharacterized YfiP protein as the SAM-dependent 3-amino-3-carboxypropyl transferase catalyzing this modification and thereby extend the list of known tRNA modification enzymes in E. coli. Similar to the Tsr3 enzymes that introduce acp modifications at U or m1Ψ nucleotides in rRNAs this protein contains a DTW domain suggesting that acp transfer reactions to RNA nucleotides are a general function of DTW domain containing proteins. The introduction of the acp3U-47 modification in E. coli tRNAs is promoted by the presence of the m7G-46 modification as well as by growth in rich medium. However, a deletion of the enzymes responsible for the modifications at position 46 and 47 in the variable loop of E. coli tRNAs did not lead to a clearly discernible phenotype suggesting that these two modifications play only a minor role in ensuring the proper function of tRNAs in E. coli.
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http://dx.doi.org/10.1093/nar/gkz1191DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026641PMC
February 2020

NAIL-MS reveals the repair of 2-methylthiocytidine by AlkB in E. coli.

Nat Commun 2019 12 6;10(1):5600. Epub 2019 Dec 6.

Department of Chemistry, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, 81377, Munich, Germany.

RNAs contain post-transcriptional modifications, which fulfill a variety of functions in translation, secondary structure stabilization and cellular stress survival. Here, 2-methylthiocytidine (msC) is identified in tRNA of E. coli and P. aeruginosa using NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) in combination with genetic screening experiments. msC is only found in 2-thiocytidine (sC) containing tRNAs, namely tRNA, tRNA, tRNA and tRNA at low abundances. msC is not formed by commonly known tRNA methyltransferases. Instead, we observe its formation in vitro and in vivo during exposure to methylating agents. More than half of the sC containing tRNA can be methylated to carry msC. With a pulse-chase NAIL-MS experiment, the repair mechanism by AlkB dependent sulfur demethylation is demonstrated in vivo. Overall, we describe msC as a bacterial tRNA modification and damage product. Its repair by AlkB and other pathways is demonstrated in vivo by our powerful NAIL-MS approach.
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http://dx.doi.org/10.1038/s41467-019-13565-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6898146PMC
December 2019

Time-resolved NMR monitoring of tRNA maturation.

Nat Commun 2019 07 29;10(1):3373. Epub 2019 Jul 29.

Expression génétique microbienne, UMR 8261, CNRS, Université de Paris, Institut de biologie physico-chimique, 13 rue Pierre et Marie Curie, 75005, Paris, France.

Although the biological importance of post-transcriptional RNA modifications in gene expression is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. Here, we introduce the application of NMR spectroscopy to observe the maturation of tRNAs in cell extracts. By following the maturation of yeast tRNA with time-resolved NMR measurements, we show that modifications are introduced in a defined sequential order, and that the chronology is controlled by cross-talk between modification events. In particular, we show that a strong hierarchy controls the introduction of the T54, Ψ55 and mA58 modifications in the T-arm, and we demonstrate that the modification circuits identified in yeast extract with NMR also impact the tRNA modification process in living cells. The NMR-based methodology presented here could be adapted to investigate different aspects of tRNA maturation and RNA modifications in general.
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http://dx.doi.org/10.1038/s41467-019-11356-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662845PMC
July 2019

Formation of tRNA Wobble Inosine in Humans Is Disrupted by a Millennia-Old Mutation Causing Intellectual Disability.

Mol Cell Biol 2019 10 11;39(19). Epub 2019 Sep 11.

Department of Biology, University of Rochester, Rochester, New York, USA

The formation of inosine at the wobble position of eukaryotic tRNAs is an essential modification catalyzed by the ADAT2/ADAT3 complex. In humans, a valine-to-methionine mutation (V144M) in ADAT3 that originated ∼1,600 years ago is the most common cause of autosomal recessive intellectual disability (ID) in Arabia. While the mutation is predicted to affect protein structure, the molecular and cellular effects of the V144M mutation are unknown. Here, we show that cell lines derived from ID-affected individuals expressing only ADAT3-V144M exhibit decreased wobble inosine in certain tRNAs. Moreover, extracts from the same cell lines of ID-affected individuals display a severe reduction in tRNA deaminase activity. While ADAT3-V144M maintains interactions with ADAT2, the purified ADAT2/3-V144M complexes exhibit defects in activity. Notably, ADAT3-V144M exhibits an increased propensity to form aggregates associated with cytoplasmic chaperonins that can be suppressed by ADAT2 overexpression. These results identify a key role for ADAT2-dependent folding of ADAT3 in wobble inosine modification and indicate that proper formation of an active ADAT2/3 complex is crucial for proper neurodevelopment.
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http://dx.doi.org/10.1128/MCB.00203-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751630PMC
October 2019

Cytosine-5 RNA methylation links protein synthesis to cell metabolism.

PLoS Biol 2019 06 14;17(6):e3000297. Epub 2019 Jun 14.

Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.
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http://dx.doi.org/10.1371/journal.pbio.3000297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594628PMC
June 2019

Benefits of stable isotope labeling in RNA analysis.

Biol Chem 2019 06;400(7):847-865

Department of Chemistry, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, D-81377 Munich, Germany.

RNAs are key players in life as they connect the genetic code (DNA) with all cellular processes dominated by proteins. They contain a variety of chemical modifications and many RNAs fold into complex structures. Here, we review recent progress in the analysis of RNA modification and structure on the basis of stable isotope labeling techniques. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the key tools and many breakthrough developments were made possible by the analysis of stable isotope labeled RNA. Therefore, we discuss current stable isotope labeling techniques such as metabolic labeling, enzymatic labeling and chemical synthesis. RNA structure analysis by NMR is challenging due to two major problems that become even more salient when the size of the RNA increases, namely chemical shift overlaps and line broadening leading to complete signal loss. Several isotope labeling strategies have been developed to provide solutions to these major issues, such as deuteration, segmental isotope labeling or site-specific labeling. Quantification of modified nucleosides in RNA by MS is only possible through the application of stable isotope labeled internal standards. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS), it is now possible to analyze the dynamic processes of post-transcriptional RNA modification and demodification. The trend, in both NMR and MS RNA analytics, is without doubt shifting from the analysis of snapshot moments towards the development and application of tools capable of analyzing the dynamics of RNA structure and modification profiles.
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http://dx.doi.org/10.1515/hsz-2018-0447DOI Listing
June 2019

Production and Application of Stable Isotope-Labeled Internal Standards for RNA Modification Analysis.

Genes (Basel) 2019 01 5;10(1). Epub 2019 Jan 5.

Department of Chemistry, Ludwig Maximilians University Munich, Butenandtstr. 5-13, 81377 Munich, Germany.

Post-transcriptional RNA modifications have been found to be present in a wide variety of organisms and in different types of RNA. Nucleoside modifications are interesting due to their already known roles in translation fidelity, enzyme recognition, disease progression, and RNA stability. In addition, the abundance of modified nucleosides fluctuates based on growth phase, external stress, or possibly other factors not yet explored. With modifications ever changing, a method to determine absolute quantities for multiple nucleoside modifications is required. Here, we report metabolic isotope labeling to produce isotopically labeled internal standards in bacteria and yeast. These can be used for the quantification of 26 different modified nucleosides. We explain in detail how these internal standards are produced and show their mass spectrometric characterization. We apply our internal standards and quantify the modification content of transfer RNA (tRNA) from bacteria and various eukaryotes. We can show that the origin of the internal standard has no impact on the quantification result. Furthermore, we use our internal standard for the quantification of modified nucleosides in mouse tissue messenger RNA (mRNA), where we find different modification profiles in liver and brain tissue.
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http://dx.doi.org/10.3390/genes10010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356711PMC
January 2019

Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides.

Nat Protoc 2019 01;14(1):283-312

Center for Integrated Protein Science Munich (CiPSM), Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.
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http://dx.doi.org/10.1038/s41596-018-0094-6DOI Listing
January 2019

Surpassing limits of static RNA modification analysis with dynamic NAIL-MS.

Methods 2019 03 3;156:91-101. Epub 2018 Nov 3.

LMU Munich, Faculty of Chemistry and Pharmacy, Department of Organic Chemistry, Butenandtstr. 5, 81377 Munich, Germany. Electronic address:

Ribonucleic acids (RNA) are extensively modified. These modifications are quantified by mass spectrometry (LC-MS/MS) to determine the abundance of a modification under certain conditions or in various genetic backgrounds. With LC-MS/MS the steady state of modifications is determined, and thus we only have a static view of the dynamics of RNA modifications. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) we overcome this limitation and get access to the dynamics of RNA modifications. We describe labeling techniques for E. coli, S. cerevisiae and human cell culture and the current instrumental limitations. We present the power of NAIL-MS but we also outline validation experiments, which are necessary for correct data interpretation. As an example, we apply NAIL-MS to study the demethylation of adenine and cytidine, which are methylated by the damaging agent methyl-methanesulfonate in E. coli. With NAIL-MS we exclude the concurrent processes for removal of RNA methylation, namely RNA degradation, turnover and dilution. We use our tool to study the speed and efficiency of 1-methyladenosine and 3-methylcytidine demethylation. We further outline current limitations of NAIL-MS but also potential future uses for e.g. relative quantification of tRNA isoacceptor abundances.
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http://dx.doi.org/10.1016/j.ymeth.2018.10.025DOI Listing
March 2019

NAIL-MS in E. coli Determines the Source and Fate of Methylation in tRNA.

Chembiochem 2018 12 20;19(24):2575-2583. Epub 2018 Nov 20.

Department of Chemistry, LMU Munich, Faculty of Chemistry and Pharmacy, Butenandtstrasse 5, 81377, Munich, Germany.

In all domains of life, the nucleobases of tRNA can be methylated. These methylations are introduced either by enzymes or by the reaction of methylating agents with the nucleophilic centers of the nucleobases. Herein, we present a systematic approach to identify the methylation sites within RNA in vitro and in vivo. For discrimination between enzymatic tRNA methylation and tRNA methylation damage in bacteria, we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS). With NAIL-MS, we clearly observed the formation of 7-methylguanosine, 3-methyluridine, and 6-methyladenosine during exposure of bacteria to the alkylating agent methyl methanesulfonate (MMS) in vivo. These damage products were not reported to form in tRNA in vivo, as they were masked by the enzymatically formed modified nucleosides in previous studies. In addition, we found formation of the known damage products 1-methyladenosine and 3-methylcytidine in vivo. With a dynamic NAIL-MS setup, we observed tRNA repair by demethylation of these two RNA modifications in vivo. Furthermore, we saw the potential repair of 6-methyladenosine but not 7-methylguanosine in bacterial tRNA.
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http://dx.doi.org/10.1002/cbic.201800525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582434PMC
December 2018

Initial single centre experience with the novel Rhythmia© high density mapping system in an all comer collective of 400 electrophysiological patients.

Int J Cardiol 2018 Dec 2;272:168-174. Epub 2018 Aug 2.

Department of Medicine I, University Hospital Munich, Ludwig Maximilians University, Munich, Germany.

Background: A novel, automatically annotating ultra-high density mapping system (Rhythmia©, Boston Scientific) collects a high number and quality of electrograms (EGMs). So far, data on general use in the electrophysiological laboratory are sparse.

Methods: We retrospectively analyzed all our ablations using Rhythmia and recorded patient clinical data, procedural parameters, and mapping parameters including the count of EGMs, mapping time, and mapping volume. Where appropriate, procedural parameters were compared over time to assess a learning curve.

Results: 400 patients underwent ablation of atrial fibrillation (n = 202), typical (n = 16) or atypical atrial flutter (n = 49), VT (n = 48), PVC (n = 35), accessory pathways (n = 14), AVNRT (n = 4), and focal atrial tachycardia (n = 32). System use was feasible, as no procedure had to be stopped for technical reasons and no ablation had to be withheld because of mapping failure, and safe, with an overall complication rate of 2.25%. Initial restrictions in manoeuvrability of the mapping catheter were overcome rapidly, as indicated by a significant decrease of fluoroscopy time (20 vs. 14 min, p = 0.02), use of contrast agent (50 vs. 40 ml; p < 0.01), and (not significant) lower procedure times (194 vs. 170 min; p = 0.12; comparing the first with the last third of patients undergoing pulmonary vein isolation only procedure). Ablation of complex left atrial, focal and ventricular tachycardias benefited from the reliable automatic annotation of a high number of EGMs.

Conclusion: The use of the Rhythmia is feasible and safe. Initial restrictions in manoeuvrability of the Orion mapping catheter were overcome rapidly. The procedures that benefit the most from ultra-high density mapping are complex left atrial tachycardias, focal tachycardias, and ventricular tachycardias.
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http://dx.doi.org/10.1016/j.ijcard.2018.07.141DOI Listing
December 2018

TRMT1-Catalyzed tRNA Modifications Are Required for Redox Homeostasis To Ensure Proper Cellular Proliferation and Oxidative Stress Survival.

Mol Cell Biol 2017 Nov 13;37(21). Epub 2017 Oct 13.

Department of Biology, Center for RNA Biology, University of Rochester, Rochester, New York, USA

Mutations in the tRNA methyltransferase 1 () gene have been identified as the cause of certain forms of autosomal-recessive intellectual disability (ID). However, the molecular pathology underlying ID-associated TRMT1 mutations is unknown, since the biological role of the encoded TRMT1 protein remains to be determined. Here, we have elucidated the molecular targets and function of TRMT1 to uncover the cellular effects of ID-causing TRMT1 mutations. Using human cells that have been rendered deficient in TRMT1, we show that TRMT1 is responsible for catalyzing the dimethylguanosine (m2,2G) base modification in both nucleus- and mitochondrion-encoded tRNAs. TRMT1-deficient cells exhibit decreased proliferation rates, alterations in global protein synthesis, and perturbations in redox homeostasis, including increased endogenous ROS levels and hypersensitivity to oxidizing agents. Notably, ID-causing TRMT1 variants are unable to catalyze the formation of m2,2G due to defects in RNA binding and cannot rescue oxidative stress sensitivity. Our results uncover a biological role for TRMT1-catalyzed tRNA modification in redox metabolism and show that individuals with TRMT1-associated ID are likely to have major perturbations in cellular homeostasis due to the lack of m2,2G modifications.
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http://dx.doi.org/10.1128/MCB.00214-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5640816PMC
November 2017

Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability.

Nat Chem Biol 2017 Aug 12;13(8):888-894. Epub 2017 Jun 12.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Genomic modification by sulfur in the form of phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, PT sulfur has redox and nucleophilic properties that suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. In experiments involving isotopic labeling coupled with mass spectrometry, we observed sulfur replacement in PTs at a rate of ∼2% h in unstressed Escherichia coli and Salmonella enterica. Whereas PT levels were unaffected by exposure to hydrogen peroxide (HO) or hypochlorous acid (HOCl), PT turnover increased to 3.8-10% h after HOCl treatment and was unchanged by HO, consistent with the repair of HOCl-induced sulfur damage. PT-dependent sensitivity to HOCl extended to cytotoxicity and DNA strand breaks, which occurred at HOCl doses that were orders of magnitude lower than the corresponding doses of HO. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during infections in humans.
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http://dx.doi.org/10.1038/nchembio.2407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5577368PMC
August 2017