tRNA fragments: novel players in intergenerational inheritance

Non-genetic inheritance is an evocative topic; in the past few years, the debate around potential inheritance of life-time experiences independent of social factors in mammals has become highly prominent due to increasing evidence for phenotypes in the offspring after paternal environmental exposures. Strikingly, two independent studies published in Science newly implicate a special class of RNA, transfer RNA fragments, in the intergenerational effects of paternal dietary intervention.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/cr.2016.2

Studies on the role of the calcium binding mitochondrial carrier Aralar in development / differentiation / survival of mesencephalic dopaminergic neurons in mice

Aralar, ein mitochondrialler Aspartat /Glutamat Transporter, zählt zur Familie der Calcium mitochonriellen carrier und ist eine grundlegende Komponente des Malat-Aspartat NADH shuttle (MAS). Das Protein ist unerlässlich für eine einwantfreie Funktion der Mitochondrien[1]. Berücksichtigt man, dass differenzierende Neuronen einen erhöhten energetischen Aufwand haben, dass diese dadurch auch mehr ROS produzieren, welche die Mitochondrien einem größerem Stress aussetzten[2] and dass die MAS shuttle Funktion eventuell einen Einfluss hat auf die Fähigkeit der Neuronen sich zu differenzieren bzw. zu überleben[3], stellt sich die Frage ob Aralar möglicherweise eine Relevanz für Zelldifferenzierung beziehungsweise Zellüberleben hat. In dieser Diplomarbeit wurde der etwaige Einfluss von Aralar auf dopaminerge Neurogenese untersucht. Unter der Verwendung von Immunohistochemie in Cryostat Schnitten des ventralen Mesencephalons (VM), Immunocytochemie in Primärzellkultur des VM und mES Zellen und Western Blot Methoden, durchgeführt in CD1 Wildtyp Mäusen konnte eine abundante Aralar Expression während kritischer Phasen dopaminerger Neurogenese bestätigt werden. Vergleichende immunohistologische and immunocytochemische Studien des VM im embryonalen Alters von E11.5, E13.5, E14.5 und E15.5 von WT(+/+) und KO (-/-) Aralar Mäusen deckten Differenzen der quantitativen Ernte an dopaminergen Neuronen (erhoben anhand TH expression) und Unterschiede der Expression des dopaminergen Transkriptionsfaktors Foxa2, wie auch des Zellcyklus Marker Ki67 und des apoptotischen Markers Casp3 auf. Eine mögliche Erklärung wäre ein Einfluss von Aralar auf die Zellüberlebenskapazität auf Grund von fehlender mitochondrieller Integrität. Darüber hinaus könnte das Abhandenkommen der MAS shuttle Aktivität metabolische Veränderungen hervorrufen und dadurch die Transkriptionskontrolle der dopaminergen Neurogenese beeinflussen. Des Weiteren wurden mES Zelllinien durch aralar (+/-) Kreuzungen erzeugt und charakterisiert. Die Pluripotenz der Linien konnte durch Immunocytochemie und Cytochemie bestätigt werden. Genotypierung, antibiotische Selektion und Western Blot Analyse zeigten jedoch, dass keine Aralar defiziente Linie unter den hergeleiteten Linien war. Daraus wurde geschlossen, dass Aralar bereits zu einem sehr frühen Zeitpunkt der Entwicklung eine Rolle spielt, vielleicht auf Grund restringierter Energiebereitstellung durch defekte Mitochondrien. Aralardefizienz im Blastocystenstadium könnte deren Detektierung erschweren.Aralar, a mitochondrial aspartate/glutamate carrier (AGC), belongs to the family of Calcium mitochondrial carriers (CaMC) and is a component of the malate-aspartate NADH shuttle (MAS). It has been shown essential for proper mitochondrial function [1]. Having in mind that differentiating neurons have increasing energetic demands, that differentiating neurons produce elevated levels of reactive oxygen species (ROS), which could expose mitochondria to an increased stress[2], and that the MAS shuttle function could be implied in differentiation and survival abilities of cells[3], the matter arises whether Aralar could be relevant for cell differentiation and/or survival capacity. In this thesis Aralar´s eventual role in dopaminergic neurogenesis was investigated. Expression studies, using immunohistochemistry of ventral mesencephalon (VM) cryostat slices, immunocytochemistry of ventral mesencephalic (VM) primary culture and mES cells and western blot methods, carried out in CD1 wildtype mice showed abundant Aralar expression during critical phases of dopaminergic neurogenesis. Comparative in vivo immunohistochemical and in vitro immunocytochemical expression studies of VM at embryonic ages E11.5, E13.5, e14.5 and E15.5 of WT(+/+) and KO (-/-) aralar mice revealed differences in dopaminergic neuron harvest (evaluated by tyrosine hydroxylase expression) and expression differences of Foxa2, a dopaminergic transcription factor, as well as the cell cycle marker Ki67 and the apoptotic marker casp3. Therefore an impact of Aralar on cell survival capacity due to missing mitochondrial integrity is suggested. Furthermore MAS shuttle activity deprivation could explain metabolic changes which could influence transcriptional control of dopaminergic neurogenesis. Moreover mES cell lines from aralar (+/-) mates have been established and characterised. Pluripotency of established lines has been approved by immunocytochemistry and cytochemistry. Still genotyping, antibiotic selection and western blot analysis demonstrated that no ara (-/-) mES cell line could be derived. This lead to the conclusion that Aralar might already be implicated at a very early point of development maybe because of restricted energy supply. Aralar deficiency at blastocyst stage might difficultate their detection

tRNA fragments: novel players in intergenerational inheritance.

Non-genetic inheritance is an evocative topic; in the past few years, the debate around potential inheritance of life-time experiences independent of social factors in mammals has become highly prominent due to increasing evidence for phenotypes in the offspring after paternal environmental exposures. Strikingly, two independent studies published in Science newly implicate a special class of RNA, transfer RNA fragments, in the intergenerational effects of paternal dietary intervention.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/cr.2016.2

Mature sperm small-RNA profile in the sparrow: implications for transgenerational effects of age on fitness.

Mammalian sperm RNA has recently received a lot of interest due to its involvement in epigenetic germline inheritance. Studies of epigenetic germline inheritance have shown that environmental exposures can induce effects in the offspring without altering the DNA sequence of germ cells. Most mechanistic studies were conducted in laboratory rodents and C.elegans while observational studies confirm the phenotypic phenomenon in wild populations of humans and other species including birds. Prominently, paternal age in house sparrows affects offspring fitness, yet the mechanism is unknown. This study provides a first reference of house sparrow sperm small RNA as an attempt to uncover their role in the transmission of the effects of paternal age on the offspring. In this small-scale pilot, we found no statistically significant differences between miRNA and tRNA fragments in aged and prime sparrow sperm. These results indicate a role of other epigenetic information carriers, such as distinct RNA classes, RNA modifications, DNA methylation and retained histones, and a clear necessity of future studies in wild populations.This work was supported by Cancer Research UK (C13474/A18583, C6946/A14492) and the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z) through E.A.M. W.M. was funded by The Nakajima Foundation and St John’s College Benefactors’ Scholarship. K.G. received funding from the Swiss National Science Foundation advanced mobility fellowship. J.S. was supported by Imperial College London

Single paternal dexamethasone challenge programs offspring metabolism and reveals multiple candidates in RNA-mediated inheritance.

Single traumatic events that elicit an exaggerated stress response can lead to the development of neuropsychiatric conditions. Rodent studies suggested germline RNA as a mediator of effects of chronic environmental exposures to the progeny. The effects of an acute paternal stress exposure on the germline and their potential consequences on offspring remain to be seen. We find that acute administration of an agonist for the stress-sensitive Glucocorticoid receptor, using the common corticosteroid dexamethasone, affects the RNA payload of mature sperm as soon as 3 hr after exposure. It further impacts early embryonic transcriptional trajectories, as determined by single-embryo sequencing, and metabolism in the offspring. We show persistent regulation of tRNA fragments in sperm and descendant 2-cell embryos, suggesting transmission from sperm to embryo. Lastly, we unravel environmentally induced alterations in sperm circRNAs and their targets in the early embryo, highlighting this class as an additional candidate in RNA-mediated inheritance of disease risk.KG was funded by the Swiss National Science Foundation early postdoc and advanced postdoc mobility a SPARK and Novartis foundation grant. Some of this work was supported by Cancer Research UK (C13474/A18583, C6946/A14492) and Wellcome (104640/Z/14/Z, 092096/Z/10/Z) to EAM. GP and MH were supported by a core grant from the Wellcome Trust. The lab of JB is currently funded by the ETH Zurich, SNSF Project Grant 310030_172889/1, ETH Research Grant ETH-20 19-1, the Kurt und Senta Herrmann-Stiftung, the Botnar Research Center for Child Health and a 3R Competence Center Project Grant. JK was supported by a Swiss-european mobility programme scholarship

Sequencing cell-type-specific transcriptomes with SLAM-ITseq.

Analysis of cell-type-specific transcriptomes is vital for understanding the biology of tissues and organs in the context of multicellular organisms. In this Protocol Extension, we combine a previously developed cell-type-specific metabolic RNA labeling method (thiouracil (TU) tagging) and a pipeline to detect the labeled transcripts by a novel RNA sequencing (RNA-seq) method, SLAMseq (thiol (SH)-linked alkylation for the metabolic sequencing of RNA). By injecting a uracil analog, 4-thiouracil, into transgenic mice that express cell-type-specific uracil phosphoribosyltransferase (UPRT), an enzyme required for 4-thiouracil incorporation into newly synthesized RNA, only cells expressing UPRT synthesize thiol-containing RNA. Total RNA isolated from a tissue of interest is then sequenced with SLAMseq, which introduces thymine to cytosine (T>C) conversions at the sites of the incorporated 4-thiouracil. The resulting sequencing reads are then mapped with the T>C-aware alignment software, SLAM-DUNK, which allows mapping of reads containing T>C mismatches. The number of T>C conversions per transcript is further analyzed to identify which transcripts are synthesized in the UPRT-expressing cells. Thus, our method, SLAM-ITseq (SLAMseq in tissue), enables cell-specific transcriptomics without laborious FACS-based cell sorting or biochemical isolation of the labeled transcripts used in TU tagging. In the murine tissues we assessed previously, this method identified ~5,000 genes that are expressed in a cell type of interest from the total RNA pool from the tissue. Any laboratory with access to a high-throughput sequencer and high-power computing can adapt this protocol with ease, and the entire pipeline can be completed in <5 d.This work was supported by grants from Cancer Research UK (C13474/A18583, C6946/A14492) and the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z) to E.A.M.; and a grant from the European Research Council (ERC-StG-338252 miRLIFE) to S.L.A. The IMP is generously supported by Boehringer Ingelheim. W.M. was supported by the Nakajima Foundation and St John’s College Benefactors’ Scholarship. K.G. was supported by a Swiss National Foundation postdoc mobility fellowship

Implication of sperm RNAs in transgenerational inheritance of the effects of early trauma in mice.

Small non-coding RNAs (sncRNAs) are potential vectors at the interface between genes and environment. We found that traumatic stress in early life altered mouse microRNA (miRNA) expression, and behavioral and metabolic responses in the progeny. Injection of sperm RNAs from traumatized males into fertilized wild-type oocytes reproduced the behavioral and metabolic alterations in the resulting offspring.We thank M. Rassoulzadegan and V. Grandjean for help with the sperm purification, F. Manuella and H. Hörster for assistance with the MSUS paradigm, H. Welzl for help with behavior, G. Vernaz for help with western blotting, R. Tweedie-Cullen and P. Nanni for help with mass spectrometry, A. Patrignani for advice on DNA and RNA quality assessment, and A. Chen and A. Brunner for constructive discussions. This work was supported by the Austrian Academy of Sciences, the University of Zürich, the Swiss Federal Institute of Technology, Roche, the Swiss National Science Foundation, and The National Center of Competence in Research “Neural Plasticity and Repair”. P.S. was supported by a Gonville and Caius College fellowship.This is the accepted manuscript. The final version is available in Nature Neuroscience 17, 667–669 (2014), doi:10.1038/nn.369

Unconventional forms of inheritance.

The discourse on hard (mendelian) versus more unconventional “soft” forms of inheritance has been blazing for long