A maize r1 gene is regulated post-transcriptionally by differential splicing of its leader

Anthocyanin biosynthesis in Zea mays is controlled by regulatory genes of the r1/b1 family that encode bHLH transcription factors. Analysis of the 381 nucleotide leader sequence of a member of this family, Sn, discloses the presence of five ATG triplets upstream of the coding region and three upstream open reading frames (uORFs) of 38, 15 and 13 amino acids respectively. RT-PCR studies revealed that a splicing event occurs in the leader region in the different tissues tested. Splicing deletes 146 nucleotides which include uORF2 and uORF3. By trans-activation experiments in maize protoplasts we find that the spliced leader, compared to the non-spliced one, reduces the number of pigmented protoplasts by four-fold. We suggest a multilevel regulation of the Sn transcription factor acting not only at the transcriptional but also at the post-transcriptional level.