Identification and analysis of dicer associated proteins in human cells

Dicer is a multidomain RNase III like enzyme involved in the initial steps of RNA interference (RNAi) and microRNA (miRNA) pathways, two mechanisms of posttranscriptional gene silencing conserved throughout most eukaryotic species. Dicer has been shown to be necessary and sufficient to cut long dsRNA and miRNA precursors into small (21-25 nt) RNAs. In metazoa, the small RNA products of Dicer are further incorporated into a multiprotein RNA induced silencing complex (RISC), which target mRNAs in a sequence specific manner to induce mRNA cleavage (guided by siRNAs) or inhibition of translation (guided by miRNAs). We aimed to identify proteins interacting with human Dicer. Specific anti- Dicer antibodies were used to immunoprecipitate Dicer from HEK293 and HeLa cells and co-immunoprecipitating proteins were analyzed by mass spectrometry. Proteins identified to specifically co-purify with Dicer fall into different categories: (1) known components of RNA silencing pathways, such as Argonaute proteins; (2) translation factors; (3) RNA helicases; (4) RNA binding proteins, and others. Among the proteins characterized by mass spectrometry we identified TRBP [HIV-1 transactivating response (TAR) RNA-binding protein], as a protein containing three dsRNA binding domains (dsRBD). We found that this protein interacts with human Dicer in vivo and in vitro, and that this interaction involves Dicer’s N-terminal helicase domain and TRBP’s third dsRBD. TRBP has previously been assigned several different functions, including inhibition of the interferon (IFN)-induced dsRNA-regulated protein kinase PKR, modulation of HIV-1 gene expression through its association with TAR, and control of cell growth. To analyze TRBP’s function in miRNA and siRNA pathways, we generated stable TRBP knock down cell lines. Using a Luciferase reporter system we showed that TRBP is necessary for efficient silencing mediated by endogenous miRNAs. Dicer cleavage activity tested in vitro using a pre-miRNA substrate was impaired in TRBP knock down extracts. However, endogenous miRNA levels were not significantly changed upon knock down of TRBP. We also found that RNAi against an endogenous gene, induced by transfection of siRNAs, was impaired in TRBP knock down cell lines. Taken together our observations show that TRBP interacts with Dicer, and suggest that, besides its impact on Dicer cleavage activity in vitro, TRBP functions mainly downstream of Dicer cleavage in miRNA and siRNA pathways. Our data provide support to the notion that large RNaseIII-type Drosha and Dicer nucleases work in conjunction with small dsRBD protein partners. They also raise the possibility of a cross talk between RNA silencing and the IFN/PKR pathways in normal and virus-infected cells. To further characterize the Dicer/TRBP complex, we generated anti-TRBP antibodies, and analyzed TRBP immunoprecipitates by mass spectrometry. Data obtained from peptide sequencing of Dicer and TRBP immunopurifications were compared, and proteins identified in both preparations, among them the E3 Ubiquitin ligase Ro52, were considered for further analyses as likely components of a Dicer/TRBP complex. Ro52, also known as Sjoegren Syndrome Antigen 1 (SSA-1) or Tripartite motif protein 21 (TRIM21) was first identified as one of the major autoantigens in Sjoegren Syndrome and Systemic Lupus Erythematosus (SLE), two severe human autoimmune diseases. Recently, it was demonstrated that Ro52 can act as a RING dependent E3 Ubiquitin ligase, and the E3 ligase activity of Ro52 was shown to be required for S-phase progression in mammalian cells. We found that Ro52 associates with the Dicer/TRBP complex, and interacts with TRBP in vitro. The presence of an E3 Ubiquitin ligase in the Dicer/TRBP complex raised the possibility that components of the RNA silencing complexes are post-translationally modified by Ubiquitin. Our data indicate that TRBP can be covalently tagged by Ubiquitin. It appears that TRBP is monoubiquitinated, rather than polyubiquitinated and degraded by the proteasome. Future work will be required to establish the function of ubiquitination of TRBP, and the role of Ro52 in RNA silencing pathways

The Relationship between Morphological Awareness and Literacy Skills in German

The objective of this dissertation was to explore relations between morphological awareness and literacy skills in German, i.e. in an orthography that is more transparent for reading than for spelling. To represent the particularly rich German morphology in the measure of morphological awareness, items covering inflection, derivation and compounding were gathered. This dissertation pursued three research questions. First, it was explored how different facets of morphological awareness relate to different literacy competencies. Because of the asymmetric orthography of German, a stronger relationship with spelling than with reading was expected. Second, it was investigated whether morphological awareness is a unique predictor of literacy skills in German after accounting for other language-related skills. Third, it was tested whether the relationship of morphological awareness with literacy skills increases with increasing literacy proficiency. Studies were conducted in different age groups. Adapted morphological awareness tasks were tested and optimised with the help of two pilot studies. Based on the findings of these two pilot studies, the cross-sectional main-study was conducted with 351 primary school children. Additionally, two exploratory studies comprising 187 university students were conducted assessing relations between morphological awareness and literacy skills in literacy competent adults. Results indicated that different facets of morphological awareness are related to literacy skills both in primary school children and in adults. Morphological awareness proved to be closer related to spelling skills than to reading skills in university students, but not in primary school children. In primary school children, phonological awareness was the key predictor of literacy skills, while morphological awareness did not have any additional predictive power. In university students, morphological awareness explained unique variance in spelling skills, but not in reading skills. In the main study with primary school children, comparisons between grade levels suggested that the relationship between morphological awareness and literacy skills does not increase with increasing literacy proficiency. However, the relative importance of morphological awareness for literacy competencies in comparison with that of phonological awareness was identified to be higher in literacy competent adults than in primary school children. Results implicate that morphological awareness is an important correlate of literacy skills in German, and that it continues to unfold its relevance beyond primary school years.In dieser Dissertation wurden Zusammenhänge zwischen der morphologischen Bewusstheit und schriftsprachlichen Fähigkeiten im Deutschen untersucht, das heißt in einer Orthographie, die für das Lesen transparenter ist als für das Schreiben. Um die morphologische Vielfalt des Deutschen bei der Messung der morphologischen Bewusstheit abbilden zu können, wurden Aufgaben zu Flexionen, Derivationen und Komposita zusammengestellt. Diese Dissertation verfolgte drei Forschungsfragen. Erstens wurde erforscht, inwiefern verschiedene Facetten der morphologischen Bewusstheit mit verschiedenen schriftsprachlichen Leistungen im Zusammenhang stehen. Aufgrund der asymmetrischen Orthographie des Deutschen wurde erwartet, dass die morphologische Bewusstheit einen engeren Zusammenhang mit Recht-schreibleistungen als mit Leseleistungen hat. Zweitens wurde getestet, ob die morphologische Bewusstheit ein zusätzlicher Prädiktor für schriftsprachliche Leistungen ist, wenn für weitere kognitive Grundlagenfertigkeiten des Schriftspracherwerbs kontrolliert wird. Drittens wurde untersucht, ob der Zusammenhang zwischen der morphologischen Bewusstheit und schrift-sprachlichen Leistungen mit zunehmenden schriftsprachlichen Fähigkeiten steigt. Die Studien wurden in verschiedenen Altersgruppen durchgeführt. Aufbauend auf den Erkenntnissen aus zwei Pilotstudien erfolgte die querschnittliche Untersuchung von 351 Grundschulkindern in der Hauptstudie. In den zwei zusätzlich durchgeführten, explorativen Studien mit insgesamt 187 Studierenden wurden Relationen zwischen der morphologischen Bewusstheit und schrift-sprachlichen Leistungen bei schriftsprachlich kompetenten Erwachsenen untersucht. Die Ergebnisse zeigten, dass verschiedene Facetten der morphologischen Bewusstheit sowohl bei Grundschulkindern als auch bei Erwachsenen mit schriftsprachlichen Fähigkeiten in Beziehung stehen. Ein engerer Zusammenhang von morphologischer Bewusstheit mit Recht-schreibfähigkeiten als mit Lesefähigkeiten konnte nur bei Erwachsenen, nicht jedoch bei Kindern festgestellt werden. In der Hauptstudie mit Grundschulkindern war die phonologische Bewusstheit der wichtigste Prädiktor für schriftsprachliche Leistungen, wobei die morphologische Bewusstheit keine zusätzliche Varianz aufklären konnte. In den Erwachsenen-studien konnte die morphologische Bewusstheit signifikant zur Varianzaufklärung von Recht-schreibleistungen, nicht jedoch von Leseleistungen beitragen. Die Ergebnisse der Hauptunter-suchung mit Schulkindern deuteten darauf hin, dass es keinen enger werdenden Zusammenhang zwischen der morphologischen Bewusstheit und schriftsprachlichen Leistungen mit zunehmender schriftsprachlicher Kompetenz gibt. Jedoch schien die relative Relevanz der morphologischen Bewusstheit für schriftsprachliche Leistungen im Vergleich zur Relevanz der phonologischen Bewusstheit bei Studierenden höher zu sein als bei Grundschulkindern. Die Ergebnisse sprechen dafür, dass die morphologische Bewusstheit eine wichtige kognitive Variable ist, die im Zusammenhang mit schriftsprachlichen Leistungen im Deutschen steht und die ihre Relevanz auch nach der Grundschulzeit weiter entfaltet

The recovery of European freshwater biodiversity has come to a halt

Owing to a long history of anthropogenic pressures, freshwater ecosystems are among the most vulnerable to biodiversity loss$^{1}$. Mitigation measures, including wastewater treatment and hydromorphological restoration, have aimed to improve environmental quality and foster the recovery of freshwater biodiversity$^{2}$. Here, using 1,816 time series of freshwater invertebrate communities collected across 22 European countries between 1968 and 2020, we quantified temporal trends in taxonomic and functional diversity and their responses to environmental pressures and gradients. We observed overall increases in taxon richness (0.73% per year), functional richness (2.4% per year) and abundance (1.17% per year). However, these increases primarily occurred before the 2010s, and have since plateaued. Freshwater communities downstream of dams, urban areas and cropland were less likely to experience recovery. Communities at sites with faster rates of warming had fewer gains in taxon richness, functional richness and abundance. Although biodiversity gains in the 1990s and 2000s probably reflect the effectiveness of water-quality improvements and restoration projects, the decelerating trajectory in the 2010s suggests that the current measures offer diminishing returns. Given new and persistent pressures on freshwater ecosystems, including emerging pollutants, climate change and the spread of invasive species, we call for additional mitigation to revive the recovery of freshwater biodiversity

The recovery of European freshwater biodiversity has come to a halt

Owing to a long history of anthropogenic pressures, freshwater ecosystems are among the most vulnerable to biodiversity loss1. Mitigation measures, including wastewater treatment and hydromorphological restoration, have aimed to improve environmental quality and foster the recovery of freshwater biodiversity2. Here, using 1,816 time series of freshwater invertebrate communities collected across 22 European countries between 1968 and 2020, we quantified temporal trends in taxonomic and functional diversity and their responses to environmental pressures and gradients. We observed overall increases in taxon richness (0.73% per year), functional richness (2.4% per year) and abundance (1.17% per year). However, these increases primarily occurred before the 2010s, and have since plateaued. Freshwater communities downstream of dams, urban areas and cropland were less likely to experience recovery. Communities at sites with faster rates of warming had fewer gains in taxon richness, functional richness and abundance. Although biodiversity gains in the 1990s and 2000s probably reflect the effectiveness of water-quality improvements and restoration projects, the decelerating trajectory in the 2010s suggests that the current measures offer diminishing returns. Given new and persistent pressures on freshwater ecosystems, including emerging pollutants, climate change and the spread of invasive species, we call for additional mitigation to revive the recovery of freshwater biodiversity.publishedVersio

A global agenda for advancing freshwater biodiversity research

Global freshwater biodiversity is declining dramatically, and meeting the challenges of this crisis requires bold goals and the mobilisation of substantial resources. While the reasons are varied, investments in both research and conservation of freshwater biodiversity lag far behind those in the terrestrial and marine realms. Inspired by a global consultation, we identify 15 pressing priority needs, grouped into five research areas, in an effort to support informed stewardship of freshwater biodiversity. The proposed agenda aims to advance freshwater biodiversity research globally as a critical step in improving coordinated actions towards its sustainable management and conservation

A global agenda for advancing freshwater biodiversity research

Global freshwater biodiversity is declining dramatically, and meeting the challenges of this crisis requires bold goals and the mobilisation of substantial resources. While the reasons are varied, investments in both research and conservation of freshwater biodiversity lag far behind those in the terrestrial and marine realms. Inspired by a global consultation, we identify 15 pressing priority needs, grouped into five research areas, in an effort to support informed stewardship of freshwater biodiversity. The proposed agenda aims to advance freshwater biodiversity research globally as a critical step in improving coordinated actions towards its sustainable management and conservation.Peer reviewe

The recovery of European freshwater biodiversity has come to a halt

Owing to a long history of anthropogenic pressures, freshwater ecosystems are among the most vulnerable to biodiversity loss1. Mitigation measures, including wastewater treatment and hydromorphological restoration, have aimed to improve environmental quality and foster the recovery of freshwater biodiversity2. Here, using 1,816 time series of freshwater invertebrate communities collected across 22 European countries between 1968 and 2020, we quantified temporal trends in taxonomic and functional diversity and their responses to environmental pressures and gradients. We observed overall increases in taxon richness (0.73% per year), functional richness (2.4% per year) and abundance (1.17% per year). However, these increases primarily occurred before the 2010s, and have since plateaued. Freshwater communities downstream of dams, urban areas and cropland were less likely to experience recovery. Communities at sites with faster rates of warming had fewer gains in taxon richness, functional richness and abundance. Although biodiversity gains in the 1990s and 2000s probably reflect the effectiveness of water-quality improvements and restoration projects, the decelerating trajectory in the 2010s suggests that the current measures offer diminishing returns. Given new and persistent pressures on freshwater ecosystems, including emerging pollutants, climate change and the spread of invasive species, we call for additional mitigation to revive the recovery of freshwater biodiversity.N. Kaffenberger helped with initial data compilation. Funding for authors and data collection and processing was provided by the EU Horizon 2020 project eLTER PLUS (grant agreement no. 871128); the German Federal Ministry of Education and Research (BMBF; 033W034A); the German Research Foundation (DFG FZT 118, 202548816); Czech Republic project no. P505-20-17305S; the Leibniz Competition (J45/2018, P74/2018); the Spanish Ministerio de Economía, Industria y Competitividad—Agencia Estatal de Investigación and the European Regional Development Fund (MECODISPER project CTM 2017-89295-P); Ramón y Cajal contracts and the project funded by the Spanish Ministry of Science and Innovation (RYC2019-027446-I, RYC2020-029829-I, PID2020-115830GB-100); the Danish Environment Agency; the Norwegian Environment Agency; SOMINCOR—Lundin mining & FCT—Fundação para a Ciência e Tecnologia, Portugal; the Swedish University of Agricultural Sciences; the Swiss National Science Foundation (grant PP00P3_179089); the EU LIFE programme (DIVAQUA project, LIFE18 NAT/ES/000121); the UK Natural Environment Research Council (GLiTRS project NE/V006886/1 and NE/R016429/1 as part of the UK-SCAPE programme); the Autonomous Province of Bolzano (Italy); and the Estonian Research Council (grant no. PRG1266), Estonian National Program ‘Humanitarian and natural science collections’. The Environment Agency of England, the Scottish Environmental Protection Agency and Natural Resources Wales provided publicly available data. We acknowledge the members of the Flanders Environment Agency for providing data. This article is a contribution of the Alliance for Freshwater Life (www.allianceforfreshwaterlife.org).Peer reviewe

A Small RNA-Based Immune System Defends Germ Cells against Mobile Genetic Elements

Transposons are mobile genetic elements that threaten the survival of species by destabilizing the germline genomes. Limiting the spread of these selfish elements is imperative. Germ cells employ specialized small regulatory RNA pathways to restrain transposon activity. PIWI proteins and Piwi-interacting RNAs (piRNAs) silence transposons at the transcriptional and posttranscriptional level with loss-of-function mutant animals universally exhibiting sterility often associated with germ cell defects. This short review aims to illustrate basic strategies of piRNA-guided defense against transposons. Mechanisms of piRNA silencing are most readily studied in Drosophila melanogaster, which serves as a model to delineate molecular concepts and as a reference for mammalian piRNA systems. PiRNA pathways utilize two major strategies to handle the challenges of transposon control: (1) the hard-wired molecular memory of prior transpositions enables recognition of mobile genetic elements and discriminates transposons from host genes; (2) a feed-forward adaptation mechanism shapes piRNA populations to selectively combat the immediate threat of transposon transcripts. In flies, maternally contributed PIWI-piRNA complexes bolster both of these lines of defense and ensure transgenerational immunity. While recent studies have provided a conceptual framework of what could be viewed as an ancient immune system, we are just beginning to appreciate its many molecular innovations

sj-docx-2-alh-10.1177_14697874221131970 – Supplemental material for The impact of feedback mode on learning gain and self-efficacy: A quasi-experimental study

Supplemental material, sj-docx-2-alh-10.1177_14697874221131970 for The impact of feedback mode on learning gain and self-efficacy: A quasi-experimental study by Christine Johannes and Astrid Haase in Active Learning in Higher Education</p