A Feedback Loop Couples Musashi-1 Activity to Omega-9 Fatty Acid Biosynthesis: A Dissertation
In bacteria, many genes involved in the biosynthesis of cofactors such as thiamine pyrophosphate (TPP) are regulated by ribo switches, regions in the 5' end of mRNAs to which the cofactor binds, thereby affecting translation and/or transcription. TPP riboswitches have now been identified in fungi, in which they alter mRNA splicing. Here, we show that addition of thiamine to cultures of the model green alga Chlamydomonas reinhardtii alters splicing of transcripts for the THI4 and THIC genes, encoding the first enzymes of the thiazole and pyrimidine branches of thiamine biosynthesis, respectively, concomitant with an increase in intracellular thiamine and TPP levels. Comparison with Volvox carteri, a related alga, revealed highly conserved regions within introns of these genes. Inspection of the sequences identified TPP riboswitch motifs, and RNA transcribed from the regions binds TPP in vitro. The THI4 riboswitch, but not the promoter region, was found to be necessary and sufficient for thiamine to repress expression of a luciferase-encoding reporter construct in vivo. The pyr1 mutant of C. reinhardtii, which is resistant to the thiamine analogue pyrithiamine, has a mutation in the THI4 riboswitch that prevents the THI4 gene from being repressed by TPP. By the use of these ribo switches, thiamine biosynthesis in C. reinhardtii can be effectively regulated at physiological concentrations of the vitamin.