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

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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.