Eukaryotic TPP riboswitch regulation of alternative splicing involving long-distance base pairing

Abstract

Thiamin pyrophosphate (TPP) riboswitches are found in organisms from all three domains of life. Examples in bacteria commonly repress gene expression by terminating transcription or by blocking ribosome binding, whereas most eukaryotic TPP riboswitches are predicted to regulate gene expression by modulating RNA splicing. Given the widespread distribution of eukaryotic TPP riboswitches and the diversity of their locations in precursor messenger RNAs (pre-mRNAs), we sought to examine the mechanism of alternative splicing regulation by a fungal TPP riboswitch from Neurospora crassa, which is mostly located in a large intron separating protein-coding exons. Our data reveal that this riboswitch uses a long-distance (∼530-nt separation) base-pairing interaction to regulate alternative splicing. Specifically, a portion of the TPP-binding aptamer can form a base-paired structure with a conserved sequence element (α) located near a 5' splice site, which greatly increases use of this 5' splice site and promotes gene expression. Comparative sequence analyses indicate that many fungal species carry a TPP riboswitch with similar intron architecture, and therefore the homologous genes in these fungi are likely to use the same mechanism. Our findings expand the scope of genetic control mechanisms relying on long-range RNA interactions to include riboswitches.

Figure 1.

Alternative splicing products of NCU01977. (A) 5′ region of precursor and alternatively spliced mRNAs for NCU01977. S1 through S4 identify alternative 5′ splice sites (GU dinucleotides) within intron 2. Gray, white and black bars represent exons, introns and TPP aptamers, respectively. AG dinucleotides represent 3′ splice sites. Arrows indicate the primer binding sites used for RT-PCR. (B) Agarose gel separation of RT-PCR products from NCU01977 mRNAs of N. crassa grown in the absence (−) or presence (+) of 30 μM thiamin. Bands I through V are DNA products from different splice variants. Asterisks denote RT-PCR products that were not sequenced. RT designates reverse transcription; M designates a lane containing DNA markers.

Figure 2.

Mutations of splice sites and their effects on TPP riboswitch regulation. (A) Design of reporter constructs with a LUC ORF fused in-frame with the natural ORF located in the exon immediately downstream of the TPP aptamer. Intron 1 has been removed. (B) LUC activities (relative light units, RLU) for WT and various alternative 5′ splice site variant (GU to GA mutations) strains. Strains were cultured in the absence (−) or presence (+) of 30 μM thiamin. Fold modulation values are the ratios of RLU values without or with thiamin supplementation. Ratio averages are the result of three independent replicates, and error bars represent standard deviation.

Figure 3.

Intron deletions reveal key regulatory elements. (A) Reporter construct design is similar to that depicted in Figure 2A with the exception that the aptamer P3 stem has been shortened (denoted by the asterisk) as depicted in Figure 4A and Supplementary Figure S3A. (B) Depiction of deletion constructs wherein the first and last nucleotides of each region deleted (horizontal lines) are provided. Shaded boxes identify regions (α and α′) that can form alternative base pairing. (C) LUC activities for WT* and various deletion constructs. Other annotations are as described in the legend to Figure 2B.

Figure 4.

Proposed mechanism of alternative splicing regulated by the pairing between α and α′. (A) Portions of α′ can alternatively base pair to form the right shoulder of the aptamer P1 stem or form a base-paired structure with α, which is closest to the 5′ splice site S1. Mutants M1 through M6 involve the shaded nucleotide variations, and their gene expression and alternative splicing activities are presented in Figure 5. (B) Sequence alignments depicting conserved nucleotides and base-pairing potential between the α and α′ elements. Boxed nucleotides can base pair between the two regions. Overlined nucleotides in α′ form the right shoulder of the aptamer P1 stem. Nucleotides depicted in gray are different from the N. crassa sequence but they retain base pairing with the distal complementary site. Ncr, N. crassa; Cgl, Chaetomium globosum; Gze, Gibberella zeae; Fgr, Fusarium graminearum; Fve, Fusarium verticillioides; Val, Verticillium albo-atrum; Vda, Verticillium dahliae; Hca, Histoplasma capsulatum; Pbr, Paracoccidioides brasiliensis; Ure, Uncinocarpus reesii; Cim, Coccidioides immitis; Cpo, Coccidioides posadasii; Mca, Microsporum canis; Mgy, Microsporum gypseum; Ptr, Pyrenophora tritici-repentis; Sno, Stagonospora nodorum; Mgr, Magnaporthe grisea.

Figure 5.

Mutational analysis of the α and α′ interaction in the NCU01977 TPP riboswitch. (A) Plot of LUC reporter gene expression for various constructs in the absence (−) or presence (+) of thiamin supplementation. Reporter constructs for Del α (deletion of the α element) and M1 through M6 (nucleotide changes depicted in Figure 4A) are based on the parent construct depicted in Figure 3A. Del G (has a deletion of a conserved G in the junction of P2 and P3 that disrupts TPP binding) and the double mutant Del α/Del G (deletion of α and G) are based on the original WT construct that retains intron 1 and the natural P3 stem. (B) Agarose gel separation of RT-PCR products from WT and all mutant constructs. Products I through IV are similar to those observed in Figure 1B, except that they are ∼200-nt shorter owing to the use of different primers.

Figure 6.

Proposed mechanism for alternative splicing control by the NCU01977 TPP riboswitch. TPP aptamers have at least two structural states: unbound when TPP is low (Top) and ligand-bound when TPP concentrations are high (Bottom). In the unbound state, α′ pairs to α, thereby bringing the otherwise distal 5′ (S1) and 3′ splice sites in proximity to favor their splicing and produce a contiguous ORF. In the TPP-bound state, the nucleotides of α′ are sequestered by the formation of a stable P1 stem, which reduces splicing at the distal 5′ splice site (S1) and favors splicing at the more proximal alternative 5′ splice sites (S2, S3 and S4).