The Role of the Lupus Autoantigen La in the Human MicroRNA Pathway

The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports correct RNA folding. In addition, La was shown to affect the biogenesis of mammalian microRNAs (miRNAs). In this study, we have analyzed the consequences of La depletion on the Argonaute (Ago)-bound small RNA (sRNA) population in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and thereby protects the cell from tRNA fragments, which might be potentially harmful by acting as unintended miRNAs. We further provide evidences, that viral non-coding RNAs (ncRNAs) perturb La’s gatekeeper function and induce the production of Ago-loaded tRNA fragments, mimicking the effects observed under La depletion. Interestingly, one specific isoleucine precursor tRNA (pre-tRNA) produces both, a tRNA and a functional miRNA, even when La is present. We demonstrate that this specific pre-tRNA is able to partially escape from La binding due to its fully complementary 5’ leader and 3’ trailer sequences. The double-stranded RNA structure of this specific isoleucine pre-tRNA reduces the affinity to La and allows processing by components of the miRNA biogenesis machinery. In sum, our study unraveled a novel aspect of La biology, showing that it supports correct pathway selection and, by that, maturation of primary Pol III transcripts. Furthermore, we characterized in molecular detail the biogenesis of a non-canonical, pre-tRNA-derived miRNA

Epstein-Barr Virus EBER Transcripts Affect miRNA-Mediated Regulation of Specific Targets and Are Processed to Small RNA Species

The oncogenic Epstein-Barr virus (EBV) expresses 44 mature microRNAs and two non-coding EBER RNAs of 167 (EBER1) and 172 (EBER2) nt length. MiRNA profiling of NK/T cell lines and primary cells and Northern blotting of EBV-infected cell lines and primary tumors revealed processing of EBER1 to short 5′-derived RNAs of approximately 23, 52 and 70 nt (EBER123, EBER152, and EBER170) and of EBER2 to 3′ fragments. The biogenesis of these species is independent of Dicer, and EBER123 does not act like a miRNA OPEN ACCESS Non-Coding RNA 2015, 1 171 to target its complementary sequence. EBER1, EBER2 and EBER123 were bound by the lupus antigen (La), a nuclear and cytoplasmic protein that facilitates RNAi. Consistent with this, the EBERs affect regulation of interleukin 1alpha (IL1α) and RAC1 reporters harboring miR target sequences, targets of miR-142-3p. However, the EBERs have no effect upon another target of miR-142-3p, ADCY9, nor on TOMM22, a target of ebv-miR-BART16, indicative of selective modulation of gene expression by the EBERs

Seed sequence polymorphism rs2168518 and allele-specific target gene regulation of hsa-miR-4513

Acknowledgements We thank Lisa Michaelis and Dr Karolina Plößl (Institute of Human Genetics, University of Regensburg) for excellent technical help and thorough proofreading of the manuscript, respectively. We thank Marina Sauer and Franz-Stephan Attenkofer (Institute of Human Genetics, University of Regensburg) for their support in generating the luciferase reporter vectors. Conflict of Interest statement. The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding German Research Foundation (GR5065/1-1 to F.G.); and the Helmut Ecker Foundation (Ingolstadt, Germany) (no. 05/17 to B.H.F.W).Peer reviewedPublisher PD

Roadmap to a One Health agenda 2030

The current fragmented framework of health governance for humans, animals and environment, together with the conventional linear approach to solving current health problems, is failing to meet today's health challenges and is proving unsustainable. Advances in healthcare depend increasingly on intensive interventions, technological developments and expensive pharmaceuticals. The disconnect grows between human health, animal health and environmental and ecosystems health. Human development gains have come with often unrecognized negative externalities affecting ecosystems. Deterioration in biodiversity and ecosystem services threatens to reverse the health gains of the last century. A paradigm shift is urgently required to de-sectoralize human, animal, plant and ecosystem health and to take a more integrated approach to health, One Health (OH). The Sustainable Development Goals (SDGs) offer a framework and unique opportunity for this. Through analysing individual SDGs, we argue the feasibility of an OH approach towards achieving them. Feasibility assessments and outcome evaluations are often constrained by sectoral politics within a national framework, historic possession of expertise, as well as tried and tested metrics. OH calls for a better understanding, acceptance and use of a broader and transdisciplinary set of assessment metrics. Key objectives of OH are presented: that humans reconnect with our natural past and accept our place in, and dependence on our planet's ecosystems; and that we recognize our dependence on ecosystem services, the impact of our development thereon and accept our responsibility towards future generations to address this. Several action points are proposed to meet these objectives.peer-reviewe

Epstein-Barr Virus EBER Transcripts Affect miRNA-Mediated Regulation of Specific Targets and Are Processed to Small RNA Species

The oncogenic Epstein-Barr virus (EBV) expresses 44 mature microRNAs and two non-coding EBER RNAs of 167 (EBER1) and 172 (EBER2) nt length. MiRNA profiling of NK/T cell lines and primary cells and Northern blotting of EBV-infected cell lines and primary tumors revealed processing of EBER1 to short 5′-derived RNAs of approximately 23, 52 and 70 nt (EBER123, EBER152, and EBER170) and of EBER2 to 3′ fragments. The biogenesis of these species is independent of Dicer, and EBER123 does not act like a miRNA OPEN ACCESS Non-Coding RNA 2015, 1 171 to target its complementary sequence. EBER1, EBER2 and EBER123 were bound by the lupus antigen (La), a nuclear and cytoplasmic protein that facilitates RNAi. Consistent with this, the EBERs affect regulation of interleukin 1alpha (IL1α) and RAC1 reporters harboring miR target sequences, targets of miR-142-3p. However, the EBERs have no effect upon another target of miR-142-3p, ADCY9, nor on TOMM22, a target of ebv-miR-BART16, indicative of selective modulation of gene expression by the EBERs

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

The Lupus Autoantigen La Prevents Mis-channeling of tRNA Fragments into the Human MicroRNA Pathway

The Lupus autoantigen La is an RNA-binding protein that stabilizes RNA polymerase III (Pol III) transcripts and supports RNA folding and has in addition been implicated in the mammalian microRNA (miRNA) pathway. Here, we have analyzed effects of La depletion on Argonaute (Ago)-bound small RNAs in human cells. We find that in the absence of La, distinct tRNA fragments are loaded into Ago proteins. Thus, La functions as gatekeeper ensuring correct tRNA maturation and protecting the miRNA pathway from potentially functional tRNA fragments. However, one specific isoleucin pre-tRNA produces both a functional tRNA and a miRNA even when La is present. We demonstrate that the fully complementary 50 leader and 30 trailer of the pre-tRNA-Ile form a double-stranded RNA molecule that has low affinity to La. Instead, Exportin-5 (Xpo5) recognizes it as miRNA precursor and transports it into the cytoplasm for Dicer processing and Ago loading

A circulating microRNA profile is associated with late-stage neovascular age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of severe vision impairment in Western populations over 55 years. A growing number of gene variants have been identified which are strongly associated with an altered risk to develop AMD. Nevertheless, gene-based biomarkers which could be dysregulated at defined stages of AMD may point toward key processes in disease mechanism and thus may support efforts to design novel treatment regimens for this blinding disorder. Circulating microRNAs (cmiRNAs) which are carried by nanosized exosomes or microvesicles in blood plasma or serum, have been recognized as valuable indicators for various age-related diseases. We therefore aimed to elucidate the role of cmiRNAs in AMD by genome-wide miRNA expression profiling and replication analyses in 147 controls and 129 neovascular AMD patients. We identified three microRNAs differentially secreted in neovascular (NV) AMD (hsa-mir-301-3p, pcorrected = 5.6*10−5, hsa-mir-361-5p, pcorrected = 8.0*10−4 and hsa-mir-424-5p, pcorrected = 9.6*10−3). A combined profile of the three miRNAs revealed an area under the curve (AUC) value of 0.727 and was highly associated with NV AMD (p = 1.2*10−8). To evaluate subtype-specificity, an additional 59 AMD cases with pure unilateral or bilateral geographic atrophy (GA) were analyzed for microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p. While we found no significant differences between GA AMD and controls neither individually nor for a combined microRNAs profile, hsa-mir-424-5p levels remained significantly higher in GA AMD when compared to NV (pcorrected<0.005). Pathway enrichment analysis on genes predicted to be regulated by microRNAs hsa-mir-301-3p, hsa-mir-361-5p, and hsa-mir-424-5p, suggests canonical TGFβ, mTOR and related pathways to be involved in NV AMD. In addition, knockdown of hsa-mir-361-5p resulted in increased neovascularization in an in vitro angiogenesis assay

An error estimate of optimal order for a nonconforming upwind finite element discretization of the Navier-Stokes equations

We analyze a finite element approximation of the stationary incompresible Navier-Stokes equations in primitive variables which is based on the nonconforming P_1/P_0 element pair of Crouzeix/Raviart and a special upwind discritization of the convective term. An optimal error estimate in a discrete H&quot;1-norm for the velocity and in the L&quot;2norm for the pressure is proved. Some numerical results are presented. (orig.)SIGLEAvailable from TIB Hannover: RR 4487(1994,13) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

The Coxiella burnetii T4SS effector protein AnkG hijacks the 7SK small nuclear ribonucleoprotein complex for reprogramming host cell transcription

Inhibition of host cell apoptosis is crucial for survival and replication of several intracellular bacterial pathogens. To interfere with apoptotic pathways, some pathogens use specialized secretion systems to inject bacterial effector proteins into the host cell cytosol. One of these pathogens is the obligate intracellular bacterium Coxiella burnetii, the etiological agent of the zoonotic disease Q fever. In this study, we analyzed the molecular activity of the anti-apoptotic T4SS effector protein AnkG (CBU0781) to understand how C. burnetii manipulates host cell viability. We demonstrate by co- and RNA-immunoprecipitation that AnkG binds to the host cell DExD box RNA helicase 21 (DDX21) as well as to the host cell 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, an important regulator of the positive transcription elongation factor b (P-TEFb). The co-immunoprecipitation of AnkG with DDX21 is probably mediated by salt bridges and is independent of AnkG-7SK snRNP binding, and vice versa. It is known that DDX21 facilitates the release of P-TEFb from the 7SK snRNP complex. Consistent with the documented function of released P-TEFb in RNA Pol II pause release, RNA sequencing experiments confirmed AnkG-mediated transcriptional reprogramming and showed that expression of genes involved in apoptosis, trafficking, and transcription are influenced by AnkG. Importantly, DDX21 and P-TEFb are both essential for AnkG-mediated inhibition of host cell apoptosis, emphasizing the significance of the interaction of AnkG with both, the DDX21 protein and the 7SK RNA. In line with a critical function of AnkG in pathogenesis, the AnkG deletion C. burnetii strain was severely affected in its ability to inhibit host cell apoptosis and to generate a replicative C. burnetii-containing vacuole. In conclusion, the interference with the activity of regulatory host cell RNAs mediated by a bacterial effector protein represent a novel mechanism through which C. burnetii modulates host cell transcription, thereby enhancing permissiveness to bacterial infection. Author summaryFor intracellular replication, Coxiella burnetii depends on a functional type IV secretion system, which is utilized to inject ~150 virulence factors, so called effector proteins, into the host cell cytosol. Activities have only been established for few of them. These effector proteins interfere with vesicular trafficking, autophagy, lipid metabolism, apoptosis, and transcription by binding and manipulating the activity of host cell proteins. Here, we report that the C. burnetii T4SS effector protein AnkG (CBU0781, Q83DF6) binds to the host cell DExD box helicase 21 (DDX21) as well as to several host cell RNAs, including the small regulatory 7SK RNA, which is an important regulator of the positive elongation factor b (pTEFb). AnkG interferes with the function of the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, leading to significant changes in host cell transcription and ensuring host cell survival. AnkG activity is essential for efficient intracellular replication of C. burnetii and its ability to inhibit apoptosis. In summary, we identified a novel process by which a bacterial effector protein manipulates the host cell for its own benefit