Thiamine biosynthesis in algae is regulated by riboswitches

Abstract

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.

Fig. 1.

TPP biosynthetic pathway in C. reinhardtii. As in all organisms, TMP is generated from the condensation of 5-hydroxyethyl-4-methylthiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate by the action of TMP synthase (THIE). In C. reinhardtii, THI4 catalyses the first committed step in the formation of the thiazole moiety from NAD⁺, glycine, and an unknown sulfur donor. The pyrimidine moiety is synthesized from aminoimidazole ribonucleotide, an intermediate in histidine and purine biosynthesis, via the action of THIC and THID. In C. reinhardtii, THID and THIE enzyme activities are found in a single, bifunctional enzyme. To generate the active cofactor, TPP, TMP is first dephosphorylated by an unknown phosphatase, and then subsequently pyrophosphorylated by thiamine pyrophosphokinase (TPK).

Fig. 2.

Identification of TPP riboswitches in the THIC and THI4 genes of C. reinhardtii. (A) The intron/exon structure of the THIC gene from C. reinhardtii below a plot showing sequence similarity between C. reinhardtii and V. carteri. Introns are depicted by thick black lines, and the exons are depicted by open rectangles. The shaded rectangle indicates an alternatively spliced exon that encodes an in-frame stop codon (indicated by an asterisk). THIC_S and THIC_L are the two alternatively spliced transcripts that are produced in a TPP-dependent manner. The red box delineates the sequence that forms the TPP riboswitch shown in C. (B) The organization of the THI4 gene from C. reinhardtii is depicted in the same manner as in A. The start codon for the functional protein is in the second exon. The two shaded rectangles indicate alternatively spliced exons, the first of which encodes a small uORF of 27 aa. THI4_S, THI4_M, and THI4_L correspond to the alternatively spliced transcripts. The red box delineates the sequence that forms the TPP riboswitch shown in D. (C) Detailed analysis of the sequence from the THIC gene that is shown in the red box in A. It can adopt the same secondary structure of stems (P) and loops (L) as TPP riboswitches and contains 20 of the 22 nucleotides that are conserved in >90% of TPP riboswitches (highlighted in red). The circled nucleotides provide the stop codon in the alternative exon. (D) Detailed analysis of the alternative exon from the THI4 gene similarly shows that it conforms to the TPP riboswitch consensus. The overlined nucleotides indicate the start codon for the uORF, and the circled nucleotides indicate the stop codon. In the pyr1 mutant of C. reinhardtii, a single base mutation alters base pairing in stem P2.

Fig. 3.

The effect of thiamine on the expression of genes containing TPP riboswitches. (A) RT-PCR showing the time course of expression of the THIC transcripts after the addition of 10 μM thiamine to C. reinhardtii cultures by using transcript-specific primers. After the addition of thiamine at time 0, the THIC_S transcript level declines, whereas that for THIC_L increases. Primers to the constitutively expressed actin gene (ACT) were used as a control. (B) Levels of thiamine (triangles) and its esters TMP (circles) and TPP (squares) in cultures of C. reinhardtii grown over the same time course. (C) RT-PCR showing the time course of expression of THI4 transcripts after the addition of 10 μM thiamine to C. reinhardtii cultures. Only THI4_S is detectable at time 0, whereas THI4_M and THI4_L appear after ≈2 h. (D) RT-PCR of THI4 transcripts in the pyr1 mutant of C. reinhardtii. THI4_S is the only transcript present throughout the time course. (E) Luciferase activity in transgenic C. reinhardtii cells expressing the THI4 riboswitch-luc construct (shown below the graph) from the constitutive PSAD promoter. Squares indicate the activity in cells grown without thiamine, and diamonds indicate the activity in cells to which 10 μM thiamine was added at t = 0.

Fig. 4.

The effect of the THI4 promoter on gene expression in vivo. RT-PCR analysis of luc transcript abundance in five independent lines of C. reinhardtii transformed with the luc gene under the control of the THI4 promoter (shown below the gel image) (A), a THI4 riboswitch-luc construct under the control of the THI4 promoter (B), or a THI4 riboswitch-luc construct under the control of the constitutive PSAD promoter (C). Cells were grown in the presence (+) or absence (−) of 10 μM thiamine. Primers were to the luc gene (LUC) or a constitutively expressed actin gene (ACT). Only those constructs with the THI4 riboswitch (B and C) showed thiamine responsiveness.

Fig. 5.

Proposed mechanism of TPP-mediated alternative splicing of the C. reinhardtii THIC transcript. (A) Diagram of the sixth intron of the THIC gene, which contains the riboswitch, showing the intron boundaries (GU/AG), the splice acceptor sites (A), and the alternatively spliced exon in red. The green lines show which regions would be joined to form lariat loops. The thick lines depict regions that can form stems. At high TPP concentrations, TPP will be bound to the riboswitch, which means that base pairing occurs between P1 and P1′. Within the intron, there are then two splice acceptor sites available, so that two splicing events occur on either side of the riboswitch. This produces the THIC_L transcript that encodes a prematurely truncated protein. (B) When there is no TPP bound to the riboswitch (as might be predicted at low intracellular TPP concentrations), it is possible for alternative base pairing to form between P1′ and the region containing the 5′ splice acceptor site (P1″), which is colored blue. There is now only one splice acceptor site available at the 3′ end of the intron, and so the entire intron is removed to form the functional THIC_S transcript. The nucleotide sequence in Fig. 2C corresponds to the sequence from P1′ to P1.