Molecular and Genetic Analysis of SHORT INTEGUMENTS1 (SIN1)

University Of Rochester, Rochester NY

Investigators

Abstract

Molecular mechanisms of cell-cell signaling in plants are poorly understood. The SHORT INTEGUMENTS1 (SIN1) gene is important for meristem function, communication between two cell layers in the ovule, and plays essential maternal and late post-zygotic roles in early embryogenesis. The SIN1 gene, containing twenty exons, instructs a 1909 amino acid long protein with a bipartite nuclear localization signal, an RNA helicase C motif, two RNase III motifs and two carboxy-terminal double stranded RNA binding motifs similar to that of the Staufen protein of Drosophila. This proposal tests models of the mechanism of function of SIN1 gene. Alternative splicing of the SIN1 mRNA will be investigated. Identification of the putative SIN1 promoter-enhancer region will be carried out by fusion of the surrounding region to reporter genes and by assaying expression of the reporters in vivo. The SIN1 protein will be localized in cells by expressing MYC-tagged (C-terminal and N-terminal tags, separately) versions of the protein followed by immunolocalization. Putative targets of SIN1 will be identified by co-immunoprecipitation of interacting RNA with anti-MYC-antibody against SIN1-MYC, followed by RT-PCR to construct a library of cDNA (in a three-hybrid vector). Members of this library will be confirmed by three-hybrid assays in yeast, and selected candidates will be sequenced. The proposed experiments will enlighten one model of a poorly investigated aspect of morphogenesis in plant, that of post-transcriptional regulation of gene expression across cellular boundaries.