By analyzing the structure of eukaryotic genes it is possible to identify two different sequence types: those that are present in the mature transcript or mRNA, called exons, and those that are not present, called introns. RNA sequences which define exon/intron boundaries, spliceosome components, and splicing factors are represented among all eukaryotes. Besides these similarities there are some differences as intron/exon size, and the number of genes coding serine/arginine -rich (SR) proteins (splicing factors). These make plants ideal systems to study evolutionary conserved strategies for alternative splicing regulation, and to find novel mechanisms.
During my PhD and postdoctoral research, we have shown that a retrograde signal generated in the chloroplast by light modulates alternative splicing. Interestingly, alternative splicing of the SR protein coding gene RS31 is severely affected. This project main goal is to identify the implicated genes, and gene products, in the light retrograde signaling pathway that affect RS31 alternative splicing. To achieve this aim I will generate a reporter construct with the coding sequence for the hygromycin phosphotransferase (Hpt), as selection marker, combined with the alternatively spliced region of RS31. Because of this reporter design, the synthesis of HPT protein would depend on the alternative splicing outcomes of RS31. Only those transgenic lines with higher amounts of alternative splicing isoforms which possess start codons (known as mRNA3 and mRNA2 for RS31) will be hygromycin-resistant. Since the abundance of these isoforms is very low in wild type plants exposed to light, these will be hygromycin sensitive in this condition, while mutants for RS31 alternative splicing would survive. By mutagenizing the transgenic lines genomes and by a further selection using hygromycin, I will be able to isolate alternative splicing regulatory factor mutants, and some of them will be involved in the retrograde signaling pathway.
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