Hammerhead Ribozymes: Functional analysis of plants motifs and establishment of an internet-based database

锤头核酶：植物基序的功能分析和基于互联网的数据库的建立

• 批准号: 224918619
• 负责人: Professor Dr. Christian Hammann
• 金额: --
• 依托单位: HMU Health and Medical University Potsdam
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/224918619?language=en
• 关键词: Hammerhead; Ribozymes; Functional; analysis; plants

The hammerhead ribozyme (HHRz) is a small catalytic RNA motif with site-specific cleavage and ligation activity that depends on divalent metal ions. In 2005, we have identified HHRz motifs in A. thaliana, as the first plant genome, and shown expression and cleavage in several tissues. Since, we could show that these motifs are conserved in several other plant genomes (and that HHRz motifs are present in a vast number of other genomes).Next to establishing an internet-based online data resource for HHRz motifs, we will investigate in this project the plant HHRz sequences in biochemical and functional detail. The Arabidopsis HHRzs are tested for tissue-specificity and sub-cellular localisation and primary and processed transcripts will be cloned. This will be functionally related to the expression of likely target mRNAs, against which the HHRzs are in antisense orientation. Based on our observation that the expression of the HHRz RNAs is influenced by light, and partially overlaps with that of the mRNAs, we will use in situ hybridisation to investigate whether the respective RNAs co-localize in the cell. Functional investigations in vivo include the analysis of T-DNA insertion lines that we are currently screening for homozygosity. Respective strains will be analysed by knock down approaches by siRNAs and ectopic RNA expression, and compared to the wild type lines.The surprising discovery of split HHRzs open the question if the HHRz might target other cellular RNAs than the afore-mentioned mRNAs in trans. To assess this possibility, we will further analyse both, T-DNA insertion lines and strains, in which we ectopically express the HHRz motifs, for changes in global expression patterns by using deep sequencing approaches. The resulting data will be correlated to wild type plants to identify potential targets of the HHRz motifs. This, in turn, will be correlated to bioinformatics analysis for the de novo prediction of possible target sequences. The temporal and spatial expression profile of thereby identified target sequences will be compared to that of the HHRzs, using the afore-mentioned methods.The enormous amount of HHRz sequences that we and other groups have uncovered in recent years makes a web-based data resource indispensable. For that purpose, will establish and host the novel and public „hammerhead ribozyme database“. This internet-based resource will not only list the motifs by organisms, but rather add all available information on genomic localisation, structure, expression, biological function etc. Upon establishment in the frame of this grant application, the associated MySQL-database will be administered in the long run by a staff member of my group to ensure their management, curation and longterm preservation for future reuse. The other three groups that have carried out searches similar to ours have agreed to grant full access to their sequences and data.

锤头状核酶（HHRz）是一种小的催化RNA基序，具有依赖于二价金属离子的位点特异性切割和连接活性。2005年，我们在A. thaliana作为第一个植物基因组，并在几个组织中表现出表达和切割。因为，我们可以证明这些基序在其他几个植物基因组中是保守的（并且HHRz基序存在于大量的其他基因组中）。接下来，我们将建立一个基于互联网的HHRz基序在线数据资源，在这个项目中，我们将调查植物HHRz序列的生化和功能细节。测试拟南芥HHRz的组织特异性和亚细胞定位，并克隆初级和加工的转录物。这将在功能上与可能的靶mRNA的表达相关，HHRz针对该靶mRNA处于反义方向。基于我们观察到HHRz RNA的表达受光的影响，并且与mRNA的表达部分重叠，我们将使用原位杂交来研究各自的RNA是否在细胞中共定位。在体内的功能研究包括T-DNA插入线，我们目前正在筛选纯合性的分析。将通过siRNA敲除方法和异位RNA表达分析相应的菌株，并与野生型菌株进行比较。分裂HHRz的惊人发现开启了HHRz是否可能靶向上述mRNA以外的其他细胞RNA的问题。为了评估这种可能性，我们将进一步分析T-DNA插入株系和菌株，其中我们异位表达HHRz基序，通过使用深度测序方法来研究全球表达模式的变化。将所得数据与野生型植物相关联以鉴定HHRz基序的潜在靶标。反过来，这将与生物信息学分析相关，用于从头预测可能的靶序列。使用上述方法，将由此鉴定的靶序列的时间和空间表达谱与HHRz序列的时间和空间表达谱进行比较。我们和其他小组近年来发现的大量HHRz序列使得基于网络的数据资源不可或缺。为此，将建立和主持新的和公共的“锤头状核酶数据库”。这个基于互联网的资源不仅会列出生物体的图案，而且会添加有关基因组定位，结构，表达，生物功能等的所有可用信息。在此资助申请的框架内建立后，相关的MySQL数据库将由我的团队的一名工作人员长期管理，以确保其管理，策展和长期保存，以供将来重用。其他三个进行了类似于我们的搜索的小组已经同意完全访问他们的序列和数据。