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

锤头状核酶(HHRz)是一种小分子催化RNA基序，其切割和连接活性依赖于二价金属离子。2005年，我们在拟南芥中发现了HHRZ基序，这是第一个植物基因组，并在几个组织中显示了表达和切割。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基序存在于大量其他基因组中)。下一步，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，我们将对植物HHRz序列进行详细的生化和功能研究。对拟南芥HHRzs进行了组织特异性和亚细胞定位测试，并将克隆原始转录本和加工转录本。这将在功能上与可能的靶标mRNAs的表达有关，HHRZ针对这些mRNAs进行反义定位。基于我们观察到HHRz RNAs的表达受到光的影响，并与mRNAs的表达部分重叠，我们将使用原位杂交来研究各自的RNAs是否在细胞内共定位。体内的功能研究包括分析我们目前正在筛选的纯合子T-DNA插入线。通过siRNAs和异位RNA表达的敲除方法对各个菌株进行分析，并与野生型品系进行比较。HHRz分裂的惊人发现开启了一个问题，即HHRz是否可能在反式中靶向上述mRNAs以外的其他细胞RNA。为了评估这种可能性，我们将进一步分析T-DNA插入线和菌株，在其中我们异位表达HHRz基序，通过使用深度测序方法，寻找全球表达模式的变化。所得数据将与野生型植物相关联，以确定HHRz基序的潜在目标。这反过来将与生物信息学分析相关联，以便从头预测可能的靶序列。我们将使用上述方法将由此确定的目标序列的时间和空间表达谱与HHRZ序列进行比较。我们和其他团队近年来发现的大量HHRZ序列使得基于网络的数据资源不可或缺。为此目的，将建立和托管小说和公共的“锤头核酶数据库”。这个基于互联网的资源不仅将按生物体列出主题，而且还将添加关于基因组定位、结构、表达、生物功能等的所有可用信息。一旦在此赠款申请框架内建立相关的MySQL数据库，相关的MySQL数据库将由我所在小组的一名工作人员进行长期管理，以确保它们的管理、管理和长期保存，以供将来重复使用。其他三个进行了与我们类似的搜索的小组已经同意完全访问他们的序列和数据。

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.