Thiamine biosynthesis in algae is regulated by riboswitches

Area of Science:

• Molecular Biology
• Algal Genetics
• Biochemistry

Background:

• Riboswitches are regulatory RNA elements controlling gene expression, primarily known in bacteria and fungi.
• Thiamine pyrophosphate (TPP) riboswitches are crucial for cofactor biosynthesis regulation.
• Previous studies identified TPP riboswitches in bacteria and fungi, affecting transcription and translation or splicing.

Purpose of the Study:

• To investigate the presence and function of TPP riboswitches in the green alga Chlamydomonas reinhardtii.
• To determine if TPP riboswitches regulate thiamine biosynthesis gene expression in algae.
• To elucidate the mechanism of TPP-mediated gene regulation in Chlamydomonas reinhardtii.

Main Methods:

• Analysis of THI4 and THIC gene transcripts in Chlamydomonas reinhardtii under varying thiamine conditions.
• Comparative sequence analysis of algal genes with known riboswitch elements.
• In vitro RNA binding assays to confirm TPP interaction with identified riboswitch motifs.
• Reporter gene assays using luciferase to assess riboswitch function in vivo.
• Characterization of a pyrithiamine-resistant mutant (pyr1) to validate riboswitch-mediated repression.

Main Results:

• Thiamine addition to Chlamydomonas reinhardtii cultures altered THI4 and THIC mRNA splicing, correlating with increased intracellular thiamine and TPP levels.
• Conserved riboswitch motifs were identified in the introns of THI4 and THIC genes in Chlamydomonas reinhardtii and Volvox carteri.
• In vitro experiments confirmed that these RNA regions bind TPP.
• The THI4 riboswitch was sufficient to confer thiamine-dependent repression of gene expression in a reporter assay.
• A mutation in the THI4 riboswitch of the pyr1 mutant abolished TPP-mediated repression.

Conclusions:

• TPP riboswitches are functional in Chlamydomonas reinhardtii, regulating thiamine biosynthesis through mRNA splicing.
• These riboswitches provide a mechanism for effective thiamine biosynthesis regulation in algae at physiological vitamin concentrations.
• The findings expand the known repertoire of riboswitch functions and their occurrence across eukaryotic lineages.