Systemic studies of RNA binding proteins in stem cell differentiation and pluripotency

What mechanisms govern and maintain cell states during the process of differentiation is a pivotal question in science. What factors govern the commitment of developmental progenitors from pluripotent stem cells is a representative example of this question. Studies of transcriptional, signaling and chromatin regulation have been highly instrumental for elucidating mechanisms pluripotency maintenance. Nevertheless, current knowledge falls short in explaining the exit from pluripotency and its coupling to lineage commitment. It is unclear how pluripotency and differentiation become stabilized in a mutually exclusive manner. Here, I deepen our knowledge concerning post-transcriptional mechanisms in pluripotency-differentiation transition. For this purpose I first characterize by quantitative mass spectrometry the changes that occur in the mRNA bound proteome (RBPome) and identify extensive dynamic rearrangements of the RBPome during early embryonic development, from naive to primed stem cell state and to purified primitive streak progenitors (Chapter I). In parallel I identified developmental post-transcriptional processing landscape and show that the dynamic mRNA binding of the RNA-binding protein TDP-43 is critical in pluripotent stem cells (PSCs) for the choice between self-renewal and differentiation/ pluripotency breakdown (Chapter II). In detail, I discovered that TDP-43 directly regulates an evolutionary conserved switch in alternative polyadenylation (APA) of hundreds of transcripts during early differentiation of mouse and human PSCs. Functional analysis revealed that TDP-43 integrates into pluripotency circuitry by repressing the production of lengthened transcripts of the pluripotency factor SOX2, which is targeted for degradation by miR-21. Furthermore, in pluripotent stem cells TDP-43 also promotes self-renewal by repressing the formation of paraspeckles, membraneless nuclear compartments found only in differentiated cells, by enhancing production of short isoform of the lncRNA NEAT1. Conversely, reduction of TDP-43 during differentiation triggers a short-to-long isoform switch of NEAT1, which polymerizes paraspeckles that in turn recruit TDP-43 and relocalise it away from its other RNA targets. Consistent with this cross-regulation, TDP-43 inhibits differentiation and improves somatic cell reprogramming, while paraspeckles promote early differentiation. These findings reveal how the exit of pluripotency is regulated by a complex posttranscriptional network, which is functionally independent from lineage choices. Apart from its role in the exit of pluripotency, this cross-regulation between paraspeckles and TDP-43 has implications in cancer and neurodegeneration

Systemic studies of RNA binding proteins in stem cell differentiation and pluripotency

What mechanisms govern and maintain cell states during the process of differentiation is a pivotal question in science. What factors govern the commitment of developmental progenitors from pluripotent stem cells is a representative example of this question. Studies of transcriptional, signaling and chromatin regulation have been highly instrumental for elucidating mechanisms pluripotency maintenance. Nevertheless, current knowledge falls short in explaining the exit from pluripotency and its coupling to lineage commitment. It is unclear how pluripotency and differentiation become stabilized in a mutually exclusive manner. Here, I deepen our knowledge concerning post-transcriptional mechanisms in pluripotency-differentiation transition. For this purpose I first characterize by quantitative mass spectrometry the changes that occur in the mRNA bound proteome (RBPome) and identify extensive dynamic rearrangements of the RBPome during early embryonic development, from naive to primed stem cell state and to purified primitive streak progenitors (Chapter I). In parallel I identified developmental post-transcriptional processing landscape and show that the dynamic mRNA binding of the RNA-binding protein TDP-43 is critical in pluripotent stem cells (PSCs) for the choice between self-renewal and differentiation/ pluripotency breakdown (Chapter II). In detail, I discovered that TDP-43 directly regulates an evolutionary conserved switch in alternative polyadenylation (APA) of hundreds of transcripts during early differentiation of mouse and human PSCs. Functional analysis revealed that TDP-43 integrates into pluripotency circuitry by repressing the production of lengthened transcripts of the pluripotency factor SOX2, which is targeted for degradation by miR-21. Furthermore, in pluripotent stem cells TDP-43 also promotes self-renewal by repressing the formation of paraspeckles, membraneless nuclear compartments found only in differentiated cells, by enhancing production of short isoform of the lncRNA NEAT1. Conversely, reduction of TDP-43 during differentiation triggers a short-to-long isoform switch of NEAT1, which polymerizes paraspeckles that in turn recruit TDP-43 and relocalise it away from its other RNA targets. Consistent with this cross-regulation, TDP-43 inhibits differentiation and improves somatic cell reprogramming, while paraspeckles promote early differentiation. These findings reveal how the exit of pluripotency is regulated by a complex posttranscriptional network, which is functionally independent from lineage choices. Apart from its role in the exit of pluripotency, this cross-regulation between paraspeckles and TDP-43 has implications in cancer and neurodegeneration

Neuronal differentiation induces SNORD115 expression and is accompanied by post-transcriptional changes of serotonin receptor 2c mRNA

The serotonin neurotransmitter system is widespread in the brain and implicated in modulation of neuronal responses to other neurotransmitters. Among 14 serotonin receptor subtypes, 5-HT2cR plays a pivotal role in controlling neuronal network excitability. Serotonergic activity conveyed through receptor 5-HT2cR is regulated post-transcriptionally via two mechanisms, alternative splicing and A-to-I RNA editing. Brain-specific small nucleolar RNA SNORD115 harbours a phylogenetically conserved 18-nucleotide antisense element with perfect complementarity to the region of 5ht2c primary transcript that undergoes post-transcriptional changes. Previous 5ht2c minigene studies have implicated SNORD115 in fine-tuning of both post-transcriptional events. We monitored post-transcriptional changes of endogenous 5ht2c transcripts during neuronal differentiation. Both SNORD115 and 5ht2c were upregulated upon neuronal commitment. We detected increased 5ht2c alternative exon Vb inclusion already at the stage of neuronal progenitors, and more extensive A-to-I editing of non-targeted sites A and B compared to adjacent adenosines at sites E, C and D throughout differentiation. As the extent of editing is known to positively correlate with exon Vb usage while it reduces receptor functionality, our data support the model where SNORD115 directly promotes alternative exon inclusion without the requirement for conversion of key adenosines to inosines, thereby favouring production of full-length receptor isoforms with higher potency

Loss of TDP-43 causes ectopic endothelial sprouting and migration defects through increased fibronectin, vcam 1 and integrin α4/β1

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4β1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development

Widespread FUS mislocalization is a molecular hallmark of amyotrophic lateral sclerosis

Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation

Loss of TDP-43 causes ectopic endothelial sprouting and migration defects through increased fibronectin, vcam 1 and integrin α4/β1

Aggregation of the Tar DNA-binding protein of 43 kDa (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia and likely contributes to disease by loss of nuclear function. Analysis of TDP-43 function in knockout zebrafish identified an endothelial directional migration and hypersprouting phenotype during development prior lethality. In human umbilical vein cells (HUVEC) the loss of TDP-43 leads to hyperbranching. We identified elevated expression of FIBRONECTIN 1 (FN1), the VASCULAR CELL ADHESION MOLECULE 1 (VCAM1), as well as their receptor INTEGRIN α4β1 (ITGA4B1) in HUVEC cells. Importantly, reducing the levels of ITGA4, FN1, and VCAM1 homologues in the TDP-43 loss-of-function zebrafish rescues the angiogenic defects indicating the conservation of human and zebrafish TDP-43 function during angiogenesis. Our study identifies a novel pathway regulated by TDP-43 important for angiogenesis during development

CLIPing the brain:studies of protein-RNA interactions important for neurodegenerative disorders

The fate of an mRNA is largely determined by its interactions with RNA binding proteins (RBPs). Post-transcriptional processing, RNA stability, localisation and translation are some of the events regulated by the plethora of RBPs present within cells. Mutations in various RBPs cause several diseases of the central nervous system, including frontotemporal lobar degeneration, amyotrophic lateral sclerosis and fragile X syndrome. Here we review the studies that integrated UV-induced cross-linked immunoprecipitation (CLIP) with other genome-wide methods to comprehensively characterise the function of diverse RBPs in the brain. We discuss the technical challenges of these studies and review the strategies that can be used to reliably identify the RNAs bound and regulated by an RBP. We conclude by highlighting how CLIP and related techniques have been instrumental in addressing the role of RBPs in neurologic diseases. This article is part of a Special Issue entitled: RNA and splicing regulation in neurodegeneration. © 2013

Policing in Slovenia

Namen prispevka: Namen prispevka je prikaz razvoja policijske dejavnosti v Sloveniji z vidika analize procesov transformacije skozi čas, s poudarkom na sodobnih trendih pluralizacije in privatizacije policijske dejavnosti po svetu in pri nas. Metode: Za namene pregleda razvoja policijske dejavnosti v Sloveniji skozi čas in trenutnega stanja so analizirani domači in tuji viri ter zakonodaja. Ugotovitve: Glavni izzivi sodobne policijske dejavnosti v Sloveniji ostajajo sodelovanje med različnimi akterji pluralne policijske družine, poenotenje standardov na področju pooblastil predstavnikov organizacij s področja policijske dejavnosti in vprašanje nadzora nad delovanjem analiziranih organizacij. Izvirnost/pomembnost prispevka: Prispevek predstavlja pregled razvoja institucij pluralne policijske družine v Sloveniji, identificira procese transformacije, pluralizacije in privatizacije policijske dejavnosti, izpostavlja sodobne izzive na področju policijske dejavnosti in opozarja na potrebnost ustreznih prilagoditev v odgovor na opisane trende razvoja v Sloveniji.Purpose: The purpose of this paper is to show the development of policing in Slovenia through a perspective of analyzing the processes of transformation over time with an emphasis on contemporary trends of pluralization and privatization of policing in our country and abroad. Design/Methods/Approach: To examine the development of policing in Slovenia over time and to overview the current state of policing, a literature review was conducted and the relevant legislation was analyzed. Findings: The main challenges of contemporary policing in Slovenia remain co-operation between the various organizations of the plural policing family, unification of standards in the field of various policing organizations’ powers, and the issue of supervision over the activities of the analyzed organizations. Originality/Value: The paper presents an overview of the development of organizations of the plural policing family in Slovenia, identifying the processes of transformation, pluralization, and privatization of policing while highlighting the contemporary challenges in the area of policing and drawing attention to the necessity of adequate responses to the analyzed trends of development in Slovenia