Interactions between proteins and RNAs underlie every aspect of plant development
and function. Accordingly, plant genomes encode >700 proteins that harbor
predicted RNA binding motifs, but few of these proteins have been studied.
To dissect RNA-protein interaction networks in plants, it is necessary to catalog
plant RNA-binding proteins, to identify the RNAs with which they interact,
and
to determine how those interactions influence the fate of the RNA and downstream
processes. This project develops tools to address each of these issues, and
applies them to a set of 40 plant-specific RNA binding proteins that function
in the
chloroplast.
Project Objectives:
- Develop a relational database of plant RNA binding proteins
(RBP) that will integrate data from rice, maize, and Arabidopsis. The database will
feature cross-referenced orthologs and paralogs, annotated with gene
models, targeting predictions, and experimental data.
- Identify RNAs associated in vivo with each of 40 chloroplast-localized
RNA binding proteins. Our approach couples coimmunoprecipitation with microarray
technology. The 40-protein set emphasizes the CRM and PPR protein families,
which are largely specific to plants and which together contain ~500 members,
most of which are predicted to be targeted to chloroplasts or mitochondria.
- Pinpoint aspects of RNA metabolism that are influenced by each
of these 40 proteins, using reverse genetics in maize and rice. Mutants will be sought
in maize through reverse genetic screening of our PML collection (see Aim
5). RNAi in rice will be used when informative mutants are not recovered
in maize.
- Test an approach to facilitate the cloning of Mu-tagged mutations
and, if successful, apply it to RNA metabolism mutants found previously. The
approach is based on the fact that most Mu-insertions that cause strong
phenotypes also cause a severe loss of mRNA from the disrupted gene, and takes advantage
of the recently released maize 70-mer microarrays (Chandler project).
- Maintain a reverse genetic screening service based on the PML
collection. Our collection of ~2300 Mu-induced chloroplast defective maize mutants, the
Photosynthetic Mutant Library (PML), serves as the basis of a reverse-genetic
screening service for genes suspected to function in chloroplast biogenesis.
- Outreach. Tutorials on rice transformation and genetics, and K-12 outreach
programs in Ithaca and Eugene.
