Initiation by a Eukaryotic RNA-dependent RNA Polymerase Requires Looping of the Template End and Is Influenced by the Template-tailing Activity of an Associated Uridyltransferase

A conserved family of eukaryotic RNA-dependent RNA polymerases (RDRs) initiates or amplifies the production of small RNAs to provide sequence specificity for gene regulation by Argonaute/Piwi proteins. RDR-dependent silencing processes affect the genotype-phenotype relationship in many eukaryotes, but the principles that underlie the specificity of RDR template selection and product synthesis are largely unknown. Here, we characterize the initiation specificity of the Tetrahymena RDR, Rdr1, as a heterologously expressed single subunit and in the context of its biologically assembled multisubunit complexes (RDRCs). Truncation analysis of recombinant Rdr1 revealed domain requirements different from those of the only other similarly characterized RDR, suggesting that there are subfamilies of the RDR enzyme with distinct structural requirements for activity. We demonstrate an apparently obligate Rdr1 mechanism of initiation in which the template end is looped to provide the hydroxyl group priming the synthesis of dsRNA. RDRC subunits with poly(U) polymerase activity can act on the template end prior to looping to increase the duplex length of product, thus impacting the small RNA sequences generated by the RDRC-coupled Dicer. Overall, our findings give new perspective on mechanisms of RDR initiation and demonstrate that non-RDR subunits of an RDRC can affect the specificity of product synthesis.     

Use, tolerance and avoidance of amplified RNA silencing by plants

In plants and several other organisms, the effects of RNA silencing can be amplified by the action of cellular RNA-DEPENDENT RNA POLYMERASES (RDRs). These enzymes were primarily studied for their role in antiviral defense in plants, but it is becoming increasingly apparent that they also have important endogenous functions, including the control of chromatin structure and the regulation of cellular gene expression. Recent evidence suggests that endogenous RDR activities intercept several RNA quality control pathways that normally prevent or restrain widespread amplification of silencing, which is likely to be detrimental. Plants appear, however, to have evolved sophisticated measures to tolerate or exploit amplified silencing under specific biological circumstances.     

Phenotype Similarity Regression for Identifying the Genetic Determinants of Rare Diseases

Rare genetic disorders, which can now be studied systematically with affordable genome sequencing, are often caused by high-penetrance rare variants. Such disorders are often heterogeneous and characterized by abnormalities spanning multiple organ systems ascertained with variable clinical precision. Existing methods for identifying genes with variants responsible for rare diseases summarize phenotypes with unstructured binary or quantitative variables. The Human Phenotype Ontology (HPO) allows composite phenotypes to be represented systematically but association methods accounting for the ontological relationship between HPO terms do not exist. We present a Bayesian method to model the association between an HPO-coded patient phenotype and genotype. Our method estimates the probability of an association together with an HPO-coded phenotype characteristic of the disease. We thus formalize a clinical approach to phenotyping that is lacking in standard regression techniques for rare disease research. We demonstrate the power of our method by uncovering a number of true associations in a large collection of genome-sequenced and HPO-coded cases with rare diseases.     

Isolation of a novel RNA-dependent RNA polymerase 6 from <Emphasis Type="Italic">Nicotiana glutinosa</Emphasis>, <Emphasis Type="Italic">NgRDR6</Emphasis>, and analysis of its response to biotic and abiotic stresses

RNA-dependent RNA polymerases (RDRs) play an important role in RNA silencing, antiviral and developmental progress. Here, we firstly isolated the full-length cDNA, genomic DNA and 5′-flanking region of RDR6 from Nicotiana glutinosa (NgRDR6). Sequences analysis revealed that the cDNA of NgRDR6 was 3,921 bp in length, and the deduced protein consisted of 1,197 amino acids, containing all highly conserved sequence motifs that are present among all RDRs families. Moreover, two introns were detected in the genomic sequences. We also firstly investigated the expression profiles of plant RDR6 under the treatments of gibberellin A (GA), H₂O_2, methyl jasmonate (MeJA), Potato virus Y (PVY), Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Rhizoctonia Solani and Colletotrichum nicotianae. In addition, the expression patterns of RDR6 in Nicotiana glutinosa under the treatments of salicylic acid (SA) and abscisic acid (ABA) were also been analyzed. The results indicated that the NgRDR6 mRNA accumulation could be induced by ABA, GA, MeJA, CMV, Rhizoctonia Solani and Colletotrichum nicotianae. In contrast, the expression level of NgRDR6 exhibited no remarkable difference under the treatments of PVY, TMV, H₂O₂ and SA. Further investigation suggested several potential cis-acting elements were found in the 5′-flanking sequence of NgRDR6, which might be responsible for the enhanced response to phytohormones.     

Biochemical activities of Arabidopsis RNA-dependent RNA polymerase 6

In Arabidopsis, genetic evidence demonstrates that RNA-dependent RNA polymerase 6 (RDR6) plays a fundamental role in at least four RNA silencing pathways whose functions range from defense against transgenes or viruses to endogene regulation in development and in stress responses. Despite its critical role in RNA silencing, the biochemical activities of RDR6 have yet to be characterized. In this study, we transiently expressed Arabidopsis RDR6 in Nicotiana benthamiana and investigated the biochemical activities of immunopurified RDR6 in vitro. We showed that RDR6 possesses terminal nucleotidyltransferase activity as well as primer-independent RNA polymerase activity on single-stranded RNAs. We found that RDR6 cannot distinguish RNAs with or without a cap or poly(A) tail. We also demonstrated that RDR6 has strong polymerase activity on single-stranded DNA. All these activities require the conserved catalytic Asp(867) residue. Our findings have important implications on the processes involving RDR6 in vivo and provide new biochemical insights into the mechanisms of RNA silencing in Arabidopsis.     

Second generation registry framework

Background

Information management systems are essential to capture data be it for public health and human disease, sustainable agriculture, or plant and animal biosecurity. In public health, the term patient registry is often used to describe information management systems that are used to record and track phenotypic data of patients. Appropriate design, implementation and deployment of patient registries enables rapid decision making and ongoing data mining ultimately leading to improved patient outcomes. A major bottleneck encountered is the static nature of these registries. That is, software developers are required to work with stakeholders to determine requirements, design the system, implement the required data fields and functionality for each patient registry. Additionally, software developer time is required for ongoing maintenance and customisation. It is desirable to deploy a sophisticated registry framework that can allow scientists and registry curators possessing standard computing skills to dynamically construct a complete patient registry from scratch and customise it for their specific needs with little or no need to engage a software developer at any stage.

Results

This paper introduces our second generation open source registry framework which builds on our previous rare disease registry framework (RDRF). This second generation RDRF is a new approach as it empowers registry administrators to construct one or more patient registries without software developer effort. New data elements for a diverse range of phenotypic and genotypic measurements can be defined at any time. Defined data elements can then be utilised in any of the created registries. Fine grained, multi-level user and workgroup access can be applied to each data element to ensure appropriate access and data privacy. We introduce the concept of derived data elements to assist the data element standards communities on how they might be best categorised.

Conclusions

We introduce the second generation RDRF that enables the user-driven dynamic creation of patient registries. We believe this second generation RDRF is a novel approach to patient registry design, implementation and deployment and a significant advance on existing registry systems.

     

Methodological aspects of estimating rare cancer prevalence in Europe: The experience of the RARECARE project

This paper describes the usage and the performance evaluation of the completeness index method in the ‘Surveillance of Rare Cancers in Europe project’ (RARECARE) for estimating rare cancer prevalence in Europe. The 15-year prevalence at 1st January 2003 for 255 cancers is obtained from a pool of 22 RARECARE cancer registries (CRs). Incidence and survival models are applied to the RARECARE database to estimate the parameters from which the completeness indices are calculated. Complete prevalence is obtained adjusting the observed 15-year prevalence by the completeness index, to account for those cancer survivors diagnosed before the CR activity started. Main factors influencing the performance of the completeness index method for rare cancers are the same as for common cancers: age distribution of incidence and lethality of the cancer. For cancers occurring in the elderly, with low survival rates and consequently a restricted number of long-term survivors we obtained completeness indices higher than 0.9. Values lower than 0.7 correspond to those cancers with good prognosis and/or incidence more concentrated at the younger ages, indicating that 15 years of follow up are insufficient to detect all prevalent cases. Validation analysis shows that for a restricted subgroup of rare cancers with very low incidence and low survival, the completeness indices were not able to adequately correct the observed prevalence even considering a registration period of 20 years. On average, sensitivity analyses show a slight overestimation of complete prevalence for rare and common cancers whose increasing incidence is known in literature. RARECARE is the largest project on rare cancers conducted to date. Improving health care programs for cancer survivors is a public health priority and prevalence data which provides important information in this field should be regularly asked to Member States and included in the EU health statistics.     

Human embryonic stem cell registries: value, challenges and opportunities

The accelerating pace of human embryonic stem cell (hESC) research has created an urgent need for the development of hESC registries, information repositories intended to gather, organize and disseminate hESC information. Although of enormous value to this evolving field, registries face significant challenges to their development. These challenges include addressing the legal and ethical issues surrounding hESC derivation as well as complex intellectual property concerns. In addition to these issues, registries must develop tools to efficiently gather, validate and present many different types of hESC information from a variety of sources. Given the pace and regulatory complexities of this field, it is important that registries develop cooperative mechanisms to avoid duplication and more efficiently support hESC research.     

Orphanet – das europäische Portal für seltene Krankheiten

Orphanet is a relational database of rare diseases and orphan drugs. This internet-based information platform was established jointly by the French Ministry of Health and the French National Institute of Health and Medical Research (INSERM) in 1997. Since 2000 Orphanet progressed as a European project. The concept was to provide all stakeholders with compiled information on rare diseases through a directory of expert services. This directory of services provides information on specialised outpatient clinics, clinical laboratories, research projects, registries, clinical trials and patient organisations from currently 38 countries. The Services are directly associated with the inventory of rare diseases; furthermore, a comprehensive encyclopaedia is available. All information is freely accessible in five languages at the website http://www.orpha.net.     

小RNA, 大本领: 22 nt siRNAs在植物适应逆境中的重要作用

RNA是传递生命遗传信息的重要介质。依据RNA是否编码蛋白质, 可分为编码RNA和非编码RNA。作为非编码RNA的核心种类之一, 小RNA在各种生命活动中均发挥重要调控作用, 其产生及功能发挥依赖于不同的DCL、RDR和AGO蛋白。目前, 植物中功能和调控方式较为明确的是以21 nt为主的miRNA和24 nt siRNA, 其它长度和类型的小RNA由于积累水平通常较低, 尚知之甚少。近日, 南方科技大学郭红卫团队发现, 拟南芥(Arabidopsis thaliana)在缺氮等逆境胁迫下可产生大量依赖于DCL2和RDR6的22 nt siRNA。22 nt siRNA与AGO1结合形成效应复合物, 抑制硝酸还原酶基因(NIA1和NIA2)等mRNA的翻译效率, 从而减少植物在营养缺失条件下的能量消耗。这意味着, 当植物遇到不利环境时, 虽然无法通过移动来逃避逆境, 但可通过诱导产生小RNA, 协调和平衡正常的生长发育与胁迫响应。     

罕见病基因治疗与孤儿药

孤儿药因面向的罕见病患者群小、市场需求低、研发成本高、缺乏政策支持等,其发展面临困境。随着精准医疗概念的提出,基 因治疗因能够从根本出发,给患者提供 “一劳永逸”的治疗,备受关注。基因治疗以单基因罕见病的治疗作为极佳切入点,为孤儿药的 研发带来了新的希望。概述基因治疗针对的疾病对象、实施策略和属性以及基因药物的结构及基因治疗的载体,以血友病的基因治疗为 例回顾罕见病基因治疗的发展,并分析罕见病基因治疗药物研发现状。