Discovery of 17 conserved structural RNAs in fungi

Abstract

Many non-coding RNAs with known functions are structurally conserved: their intramolecular secondary and tertiary interactions are maintained across evolutionary time. Consequently, the presence of conserved structure in multiple sequence alignments can be used to identify candidate functional non-coding RNAs. Here, we present a bioinformatics method that couples iterative homology search with covariation analysis to assess whether a genomic region has evidence of conserved RNA structure. We used this method to examine all unannotated regions of five well-studied fungal genomes (Saccharomyces cerevisiae, Candida albicans, Neurospora crassa, Aspergillus fumigatus, and Schizosaccharomyces pombe). We identified 17 novel structurally conserved non-coding RNA candidates, which include 4 H/ACA box small nucleolar RNAs, 4 intergenic RNAs, and 9 RNA structures located within the introns and untranslated regions (UTRs) of mRNAs. For the two structures in the 3′ UTRs of the metabolic genes GLY1 and MET13, we performed experiments that provide evidence against them being eukaryotic riboswitches.

RNA structure landscape ofS. cerevisiaeintrons

“…Covariation across sequence alignments can identify functional secondary structures by identifying signals for compensatory mutations across related species. We evaluated DMS support for structures with covarying residues identified through R-scape by Gao, et al (2021). 19 To supplement this set of structures with predicted covariation, we followed this approach to scan for covariation in S. cerevisiae introns across the Ascomycota phylum, requiring the presence of at least one significant covarying pair in a stem of at least three base-pairs (more stringent cutoffs were used in Gao, et al 2021 19 ).…”

“…We evaluated DMS support for structures with covarying residues identified through R-scape by Gao, et al (2021). 19 To supplement this set of structures with predicted covariation, we followed this approach to scan for covariation in S. cerevisiae introns across the Ascomycota phylum, requiring the presence of at least one significant covarying pair in a stem of at least three base-pairs (more stringent cutoffs were used in Gao, et al 2021 19 ). We focused on the 11 introns with length at least 200 nucleotides where covarying residues were consistent with the predicted minimum free energy structure (Fig.…”

“…Beyond snoRNA-containing introns, covarying residues were identified in four introns from our covariation scan and in two introns from Gao, et al (2021). 19 First, we identified covariation in stems of RPL7A’s first intron, and these covarying residues were included in high confidence stems forming a 3-way junction (Fig. 3D).…”

“…Scans for covariation can provide candidate functional structures maintained through evolution. Covariation has been pinpointed thus far in a handful of yeast introns (RPS9A, 7 RPS9B, RPS13, 19 RPL7B, 19 and RPL7A). However, some functional structures in S. cerevisiae introns may be missed due to the limited power of existing sequence alignments for identifying significantly covarying residues, 51 with high sequence variation and large-scale deletions often present between aligned intron sequences.…”

“…Without structural data, it is challenging to assess the ways introns fold in vivo for specific cases, and it is difficult to understand any global structural features that might participate in splicing regulation. Though scans for covariation across sequence alignments have been able to identify potentially functional intron structures, 19 signals for covariation are limited by high sequence divergence in intronic regions, and functional structures from some prior case studies have not been identified by these scans. [16][17][18]20 In vivo chemical probing approaches provide an avenue for obtaining necessary structural data on RNA across the transcriptome.…”