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 four H/ACA box small nucleolar RNAs, four intergenic RNAs and nine 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.

Thirteen dubious ways to detect conserved structural RNAs

“…And additional screens starting from four different fungi species (C. albicans, N. crassa, A. fumigatus, and S. pombe) expanding three different Ascomycota subphyla resulted in the discovery of different structural RNAs which were not found in the other subphyla. 47 As vertebrate examples, the IREs and SECIS structural RNAs both present in the pufferfish positive control did not report any homologs in an nhmmer search performed with E-values <1 Â 10 À10 . In addition, using the UTR of the human GPX1 gene that includes a SECIS structure, an nhmmer search against a vertebrate database tuned to the evolutionary divergence of the structural RNaseP RNA, and using larger E-values <1 results in an alignment of 156 vertebrate sequences with 61% identity, but it does not report any covarying base pairs (alignment is provided in the supplemental material).…”

“…We show control screens in which a set of known structural RNAs in some genome ( S. cerevisiae and T. rubripes ) are used to search a genomic database with a homology method (nhmmer) to find homologs, build an alignment and evaluate the potential for a conserved RNA structure with R‐scape. (a) Control screen of 80 known structural ncRNAs in the budding yeast S. cerevisiae using a database of 1,371 complete fungal genomes 47 . Each RNA is represented by the observed number of significantly covarying base pairs versus the expected number of covarying pairs.…”

“…This method has controllable specificity as we showed in a recent analysis of novel ncRNAs in fungi, where we identified 21 new structural RNAs, 17 of which are novel, with an estimated specificity of 0.003 total false positives in a search over 134,000 alignments of conserved noncoding genomic regions, and 78% (62/80) sensitivity to detect known structural RNAs in S. cerevisiae. 47 Controversy still remains over lncRNAs and how to decide which have biologically relevant structures. In our experience so far using R-scape at the genome scale, we have observed a variety of important sources of confounding covariation.…”

“…For the 4,826 annotated intronless protein-coding genes in S. cerevisiae, we generated alignments using nhmmer 93 (E-value <1.0 Â 10 À10 ) searches of a database of 1,371 genomes of the Ascomycota fungal phylum. 47 (a) The observed fraction of codons for which the c1-c2 (or c1-c3 or c2-c3) codon positions significantly covary (R-scape E-value <0.05) conditioned on the corresponding amino acid (as represented by the S. cerevisiae aligned codon). (b) Comparison of the observed withincodon covariation per amino acid to the expected covariation (measured by MI) given the model of evolution.…”

Advances in Non-Coding RNA Sequencing

“…Recently, RNA sequencing has been used to assess the effect of ncRNA deletion on gene expression in yeast [ 212 ], as well as the discovery of 17 novel structural ncRNAs conserved amongst five fungal species [ 216 ]. Another highlight is the discovery of a positive lncRNA regulator of carotenoid production in fungi of the genus Fusarium [ 217 ].…”