Expression profiling of transgenic Arabidopsis over- and underexpressing SR45, a plant-specific serine/arginine-rich factor

Accurate pre-mRNA splicing is of paramount importance to all organisms. Though the basic steps of the introns splicing process are similar across yeast, mammals and plants, the repertoire of splicing factors involved in this regulated event vary substantially. The presence of several plant-specific splicing factors, and the fact that most plant introns are not excised accurately in mammalian splicing extracts and vice versa, lend support to the specific splice site mechanisms operating in these different organisms.; Arabidopsis SR45 is a plant-specific Ser/Arg-rich splicing factor that is known to interact with U1-70K, which is bound to the U1 snRNA and needed for recognition of 5' splice sites. This makes it an excellent candidate for a splicing factor involved in early intron recognition. To evaluate the effects of AtSR45 overexpression on the constitutive and alternative splicing pattern of other transcripts, crosses were generated between transgenic Arabidopsis lines overexpressing mGFP-SR45 and wtGFP transcripts. RT-PCR and Northern analyses of the F1 generation of these crosses revealed a surprising degree of variation in the SR45 transcript levels compared to constant wtGFP transcript levels and led to the initial suggestions that transgene-induced silencing caused the depletion of mGFP-SR45 transcripts. Subsequent RT-PCR analyses at different time points in plant growth verified this suggestion and demonstrated that silencing of mGFP-SR45 transcripts is developmentally controlled commencing at fifth-sixth week and reset during seed development. Genomic Southern analyses revealed that the transgene coding region was hypermethylated and that the extent of methylation progressed with age. Additional analyses indicated that the silencing of the mGFP-SR45 transcript was transitive, capable of systemic movement throughout the plant and capable of cosuppressing endogenous SR45 transcripts. Together, these characteristics indicated that the mGFP-SR45 transcripts are post-transcriptionally degraded.; The effects of SR45 depletion on other transcripts have been studied using RT-PCR analyses and more recently using oligoarray approaches. In plants completely silenced for the transgenic mGFP-SR45 transcript and nearly completely silenced for the endogenous SR45 transcript, several other splicing factor transcripts are downregulated or alternatively spliced. Global oligoarray profiling of an initial set of plants overexpressing or underexpressing SR45 transcripts has indicated that SR45 depletion downregulates a number of important transport factors (nucleoporin 155, exportin 1) as well as several stress-related transcripts that may represent secondary downstream targets. As a result of these secondary effects, it is likely that SR45-silenced plants are more susceptible to stress.