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.

Genome-Wide Analysis of RNA Secondary Structure

“…In the fungus Neurospora crassa, TPP binding favors a long-distance (>500 nucleotides distant) base-pairing interaction that loops out internal 5 splice sites, thereby favoring a specific alternative splicing configuration (58). In contrast to the approximately two dozen experimentally validated classes of riboswitches in prokaryotes (9), the TPP riboswitch is the only known eukaryotic riboswitch identified to date, but high-throughput screening for metabolite effects on RNA structuromes may identify eukaryotic RNA sequences that perform as functional riboswitches, perhaps with the involvement of proteins.…”

IRBIS: a systematic search for conserved complementarity

“…Nevertheless, a novel direction in which MSA-based methods could next develop is the exhaustive genome-wide analysis of short, well-conserved intronic motifs. In the majority of studied cases, the evolutionary constraints were so high that the complementary motifs remained almost unchanged during large evolutionary distances such as, for instance, mammalian radiation (Raker et al 2009;Pervouchine et al 2012;Li and Breaker 2013). Therefore, the computational tools that make use of compensatory base changes, in fact, cannot use this information because mutations in base-paired regions do not occur frequently enough.…”

“…Recent studies on RSSs in eukaryotic genes revealed widespread occurrence of long-range RRI with diverse functions such as riboswitches (Li and Breaker 2013) and mediators of exon skipping (Lovci et al 2013), mutually exclusive exon choice (Kreahling and Graveley 2005;Yang et al 2011), and other types of alternative splicing events (Raker et al 2009;Pervouchine et al 2012). Many of these structures are located in regions lacking reliable sequence alignments and contain long, ultraconserved stretches of complementary nucleotides, in which the interacting bases can be separated by distances as large as 10 kb.…”

Computational Analysis of Conserved RNA Secondary Structure in Transcriptomes and Genomes

“…Another view is that many supposed noncoding transcripts are the result of experimental artifacts, analysis errors, and transcriptional noise (4,99,108). On the one hand, the repertoire of functions for RNA certainly continues to expand, for noncoding RNA transcripts (2,39,40,83,118), for cis-regulatory RNA sequences in messenger RNAs (32,42,44,80,84,92), and for catalytic RNAs (74,90). On the other hand, high-throughput experiments and computational analysis pipelines have been found to suffer from serious systematic artifacts (65,108,132), and RNA biogenesis, like any biochemical process, must have some background level of infidelity (75,99).…”