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  • 1
    Language: English
    In: Clinical kidney journal, February 2017, Vol.10(1), pp.27-29
    Description: Interest in microRNAs (miRNAs) has dramatically increased in recent years not only because they regulate mRNA expression, and thus many physiological or pathophysiological processes, but also because they could serve as biomarkers. Next to analysis of tissue miRNA expression, measurement in body fluids such as blood or urine is attractive because miRNA in microvesicles or bound to protein is very stable. Currently it is unclear whether these circulating miRNAs are tissue and disease specific or represent more general pathologies like inflammation. In addition pre-analytical sample handling and variable analysis techniques affect the results and thus much more work needs to be done before one can draw a final conclusion about their clinical utility.
    Keywords: Analysis Technique ; Biomarker ; Circulating Microrna ; Kidney Function ; Renal Disease
    ISSN: 2048-8505
    E-ISSN: 20488513
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  • 2
    Article
    Article
    Language: English
    In: RNA (New York, N.Y.), April 2015, Vol.21(4), pp.645-7
    Description: In the late eighties, I became interested in non-protein-coding RNAs (ncRNAs) during the completion of my PhD in Munich (Germany) and continued my interest, as a postdoc, in one of the main centers for ribosomal RNA and protein synthesis research, the lab of Harry Noller at UCSC in California (USA). Shortly after I arrived at Harry's lab, he published one of his hallmark papers in Science, demonstrating that ribosomal RNA, mainly devoid of ribosomal proteins, was able to catalyze protein synthesis. This finding implied a catalytic function for ribosomal RNA rather than a scaffolding function. Many of his former students and postdocs, me included, still regret that Harry didn't receive the Nobel prize for his pioneering work on ribosomes. After all, his lab was the first to sequence the 16S and 23S RNAs (rRNAs) from E. coli, proposed the first 2D and 3D structure models of rRNAs and the ribosome, and also investigated, by chemical probing, its interaction with tRNA and antibiotics. In addition, he was the first to publish the crystal structure of the 70S ribosome. I know many scientists who would not have taken it lightly, when they missed the Nobel prize by such a small margin, as Harry probably did. However, when I once asked Harry whether he would like to receive the Nobel Prize at some point in his career, he answered: “Hell no, I would rather know how the ribosome really works…”.
    Keywords: Transcriptome ; RNA -- Genetics
    ISSN: 13558382
    E-ISSN: 1469-9001
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 16 January 2018, Vol.115(3), pp.E382-E389
    Description: Termination of protein synthesis is triggered by the recognition of a stop codon at the ribosomal A site and is mediated by class I release factors (RFs). Whereas in bacteria, RF1 and RF2 promote termination at UAA/UAG and UAA/UGA stop codons, respectively, eukaryotes only depend on one RF (eRF1) to initiate peptide release at all three stop codons. Based on several structural as well as biochemical studies, interactions between mRNA, tRNA, and rRNA have been proposed to be required for stop codon recognition. In this study, the influence of these interactions was investigated by using chemically modified stop codons. Single functional groups within stop codon nucleotides were substituted to weaken or completely eliminate specific interactions between the respective mRNA and RFs. Our findings provide detailed insight into the recognition mode of bacterial and eukaryotic RFs, thereby revealing the chemical groups of nucleotides that define the identity of stop codons and provide the means to discriminate against noncognate stop codons or UGG sense codons.
    Keywords: Mrna Modification ; Peptide Release ; Release Factor ; Ribosome ; Translation ; Codon, Terminator -- Genetics ; Escherichia Coli -- Metabolism ; Peptide Termination Factors -- Physiology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    In: Nature Protocols, 2011, Vol.6(2), p.166
    Description: Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.
    Keywords: Genomic Libraries -- Usage ; Rna Synthesis -- Research ; Dna Sequencing -- Methods;
    ISSN: 1754-2189
    E-ISSN: 17502799
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  • 5
    Language: English
    In: Nucleic acids research, July 2012, Vol.40(13), pp.6001-15
    Description: Protein-coding genes, guiding differentiation of ES cells into neural cells, have extensively been studied in the past. However, for the class of ncRNAs only the involvement of some specific microRNAs (miRNAs) has been described. Thus, to characterize the entire small non-coding RNA (ncRNA) transcriptome, involved in the differentiation of mouse ES cells into neural cells, we have generated three specialized ribonucleo-protein particle (RNP)-derived cDNA libraries, i.e. from pluripotent ES cells, neural progenitors and differentiated neural cells, respectively. By high-throughput sequencing and transcriptional profiling we identified several novel miRNAs to be involved in ES cell differentiation, as well as seven small nucleolar RNAs. In addition, expression of 7SL, 7SK and vault-2 RNAs was significantly up-regulated during ES cell differentiation. About half of ncRNA sequences from the three cDNA libraries mapped to intergenic or intragenic regions, designated as interRNAs and intraRNAs, respectively. Thereby, novel ncRNA candidates exhibited a predominant size of 18-30 nt, thus resembling miRNA species, but, with few exceptions, lacking canonical miRNA features. Additionally, these novel intraRNAs and interRNAs were not only found to be differentially expressed in stem-cell derivatives, but also in primary cultures of hippocampal neurons and astrocytes, strengthening their potential function in neural ES cell differentiation.
    Keywords: Cell Differentiation -- Genetics ; Embryonic Stem Cells -- Metabolism ; Neural Stem Cells -- Metabolism ; RNA, Untranslated -- Metabolism
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 6
    Language: English
    In: Nucleic acids research, 29 January 2016, Vol.44(2), pp.852-62
    Description: Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m(6)A), 5-methylcytosine (m(5)C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m(5)C and m(6)A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation. We introduced m(5)C, m(6)A, Ψ or 2'-O-methylated nucleotides at each of the three positions within a codon of the bacterial ErmCL mRNA and analyzed their influence on translation. Depending on the respective nucleotide modification, as well as its position within a codon, protein synthesis remained either unaffected or was prematurely terminated at the modification site, resulting in reduced amounts of the full-length peptide. In the latter case, toeprint analysis of ribosomal complexes was consistent with stalling of translation at the modified codon. When multiple nucleotide modifications were introduced within one codon, an additive inhibitory effect on translation was observed. We also identified the m(5)C modification to alter the amino acid identity of the corresponding codon, when positioned at the second codon position. Our results suggest a novel mode of gene regulation by nucleotide modifications in bacterial mRNAs.
    Keywords: Adenosine -- Analogs & Derivatives ; Pseudouridine -- Genetics ; RNA, Bacterial -- Genetics ; RNA, Messenger -- Genetics
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 7
    Language: English
    In: Nucleic acids research, June 2010, Vol.38(10), pp.e113
    Description: Up to 450,000 non-coding RNAs (ncRNAs) have been predicted to be transcribed from the human genome. However, it still has to be elucidated which of these transcripts represent functional ncRNAs. Since all functional ncRNAs in Eukarya form ribonucleo-protein particles (RNPs), we generated specialized cDNA libraries from size-fractionated RNPs and validated the presence of selected ncRNAs within RNPs by glycerol gradient centrifugation. As a proof of concept, we applied the RNP method to human Hela cells or total mouse brain, and subjected cDNA libraries, generated from the two model systems, to deep-sequencing. Bioinformatical analysis of cDNA sequences revealed several hundred ncRNP candidates. Thereby, ncRNAs candidates were mainly located in intergenic as well as intronic regions of the genome, with a significant overrepresentation of intron-derived ncRNA sequences. Additionally, a number of ncRNAs mapped to repetitive sequences. Thus, our RNP approach provides an efficient way to identify new functional small ncRNA candidates, involved in RNP formation.
    Keywords: Gene Library ; RNA, Untranslated -- Metabolism ; Ribonucleoproteins -- Chemistry
    ISSN: 03051048
    E-ISSN: 1362-4962
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  • 8
    Language: English
    In: Biological Psychiatry, 15 June 2017, Vol.81(12), pp.979-989
    Description: MicroRNA (miRNA)-mediated control of gene expression suggests that miRNAs are interesting targets and/or biomarkers in the treatment of anxiety- and trauma-related disorders, where often memory-associated gene expression is adversely affected. The role of miRNAs in the rescue of impaired fear extinction was assessed using the 129S1/SvlmJ (S1) mouse model of impaired fear extinction. miRNA microarray analysis, reverse transcription polymerase chain reaction, fluorescent in situ hybridization, lentiviral overexpression, and Luciferase reporter assays were used to gain insight into the mechanisms underlying miRNA-mediated normalization of deficient fear extinction. Rescuing impaired fear extinction via dietary zinc restriction was associated with differential expression of miRNAs in the amygdala. One candidate, miR-144-3p, robustly expressed in the basolateral amygdala, showed specific extinction-induced, but not fear-induced, increased expression in both extinction-rescued S1 mice and extinction-intact C57BL/6 (BL6) mice. miR-144-3p upregulation and effects on subsequent behavioral adaption was assessed in S1 and BL6 mice. miR-144-3p overexpression in the basolateral amygdala rescued impaired fear extinction in S1 mice, led to enhanced fear extinction acquisition in BL6 mice, and furthermore protected against fear renewal in BL6 mice. miR-144-3p targets a number of genes implicated in the control of plasticity-associated signaling cascades, including , , and . In functional interaction studies, we revealed that the miR-144-3p target, PTEN, colocalized with miR-144-3p in the basolateral amygdala and showed functional downregulation following successful fear extinction in S1 mice. These findings identify a fundamental role of miR-144-3p in the rescue of impaired fear extinction and suggest this miRNA as a viable target in developing novel treatments for posttraumatic stress disorder and related disorders.
    Keywords: Anxiety- and Trauma-Related Disorders ; Basolateral Amygdala, Fear ; Micrornas ; Pi3k/Akt ; Signaling Cascade Modulation ; Medicine ; Biology ; Chemistry
    ISSN: 0006-3223
    E-ISSN: 1873-2402
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  • 9
    Language: English
    In: Psychoneuroendocrinology, September 2017, Vol.83, pp.82-82
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.psyneuen.2017.07.457 Byline: Nicolas Singewald, V. Maurer, M. Oberhauser, A. Wille, A. Lusser, S.B. Sartori, A. Sah, R. Gstir, A. HAaAaAeA ttenhofer, F. Sananbenesi, Fischer, T. Bredy, N. Whittle, C. Murphy Author Affiliation: Department of Pharmacology and Toxicology, Institute of Pharmacy and CMBI, Univ Innsbruck, Austria
    Keywords: Medicine ; Anatomy & Physiology
    ISSN: 0306-4530
    E-ISSN: 1873-3360
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  • 10
    Language: English
    In: The American Journal of Human Genetics, 2010, Vol.87(6), pp.802-812
    Description: The genetic etiology of prostate cancer, the most common form of male cancer in western countries, is complex and the interplay of disease genes with environmental factors is far from being understood. Studies on somatic mitochondrial DNA (mtDNA) mutations have become an important aspect of cancer research because these mutations might have functional consequences and/or might serve as biosensors for tumor detection and progression. We sequenced the entire mitochondrial genome (16,569 bp) from 30 prospectively collected pairs of macrodissected cancerous and benign cells from prostate cancer patients and compared their genetic variability. Given recent concerns regarding the authenticity of newly discovered mtDNA mutations, we implemented a high-quality procedure for mtDNA whole-genome sequencing. In addition, the mitochondrial genes , , , and were sequenced in further 35 paired samples from prostate cancer patients. We identified a total of 41 somatic mutations in 22 out of 30 patients: the majority of these mutations have not previously been observed in the human phylogeny. The presence of somatic mutations in transfer RNAs (tRNAs) was found to be associated with elevated PSA levels (14.25 ± 5.44 versus 7.15 ± 4.32 ng/ml; p = 0.004). The level and degree of heteroplasmy increased with increasing tumor activity. In summary, somatic mutations in the mitochondrial genome are frequent events in prostate cancer. Mutations mapping to mitochondrial tRNAs, ribosomal RNAs, and protein coding genes might impair processes that occur within the mitochondrial compartment (e.g., transcription, RNA processing, and translation) and might finally affect oxidative phosphorylation.
    Keywords: Biology
    ISSN: 0002-9297
    E-ISSN: 1537-6605
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