Poster: Late and Moved Abstracts

Abs # 977: Developmental silencing and gene knockout analysis of the ser/arg-rich splicing factor SR45 in Arabidopsis thaliana

Presenter:	balasubramanian, dheepa , dbalasub@uiuc.edu
Authors	balasubramanian, dheepa  (A)   kronforst, John W (A)   Reddy, A.S.N  (B)   Schuler, Mary A (A)
Affiliations:	(A): university of illinois (B): Colorado State University

SR45 is a plant-specific splicing factor protein originally identified by yeast two-hybrid analysis to binding to U1-70K, a U1 snRNP protein, and SR33, a plant specific Ser/Arg-rich protein related to human SC35 (Golovkin and Reddy, 1999). This interaction of SR45 with U1-70K, which is situated at the 5กฏ splice site of introns during the early stages of intron splicing, suggested that this protein functions in the process of plant intron recognition. The structure of SR45 is unique in that it contains two SR domains separated by a single RBD where the SR domains are used in protein-protein interactions and the RBD domains bind RNA. Overexpression analysis of the SR45 protein attempted by crossing transgenic parental mGFP-SR45 lines to transgenic wtGFP lines resulted in developmental silencing of the mGFP-SR45 transgene beginning at 4 weeks and progressing as plants mature. Genomic DNA methylation analysis and GFP fluorescence analysis are consistent with silencing being mediated at a non-heritable post-transcriptional level. Analysis of the effects that this silencing has on other alternatively and constitutively spliced transcripts indicates that mGFP-SR45 transcript depletion generates different types of downstream effects. Analysis of a T-DNA knockout line (SALK_063528) carrying an insertion immediately upstream from the transcription start site of the SR45 gene depletes endogenous SR45 transcripts and generates the same classes of downstream effects as seen in SR45 silenced plants. We are currently analyzing the range of transcripts affected by this depletion. Golovkin, M. and Reddy, A.S.N. (1999) J. Biol. Chem. 274, 36428-36438.