Conserved secondary structures in Aspergillus

Abstract

Background: Recent evidence suggests that the number and variety of functional RNAs (ncRNAs as well as cis-acting RNA elements within mRNAs) is much higher than previously thought; thus, the ability to computationally predict and analyze RNAs has taken on new importance. We have computationally studied the secondary structures in an alignment of six Aspergillus genomes. Little is known about the RNAs present in this set of fungi, and this diverse set of genomes has an optimal level of sequence conservation for observing the correlated evolution of base-pairs seen in RNAs.

Methodology/principal findings: We report the results of a whole-genome search for evolutionarily conserved secondary structures, as well as the results of clustering these predicted secondary structures by structural similarity. We find a total of 7450 predicted secondary structures, including a new predicted approximately 60 bp long hairpin motif found primarily inside introns. We find no evidence for microRNAs. Different types of genomic regions are over-represented in different classes of predicted secondary structures. Exons contain the longest motifs (primarily long, branched hairpins), 5' UTRs primarily contain groupings of short hairpins located near the start codon, and 3' UTRs contain very little secondary structure compared to other regions. There is a large concentration of short hairpins just inside the boundaries of exons. The density of predicted intronic RNAs increases with the length of introns, and the density of predicted secondary structures within mRNA coding regions increases with the number of introns in a gene.

Conclusions/significance: There are many conserved, high-confidence RNAs of unknown function in these Aspergillus genomes, as well as interesting spatial distributions of predicted secondary structures. This study increases our knowledge of secondary structure in these aspergillus organisms.

Figure 1. Obtaining predicted secondary structures and structural classes.

Overlapping search window hits are grouped into predicted secondary structures. Since most predicted secondary structures are primarily contained within a single search window, we clustered search window hits by structural similarity into structural classes.

Figure 2. Examples of predicted secondary structure motifs by region of genome.

a) Examples of long, branched hairpins found in exonic regions; b) New bulgy hairpin motif found in intronic regions; c) Examples of known or predicted noncoding RNAs found in intergenic regions; d) Examples of short hairpins found in 5′ UTR regions; e) Examples of short hairpins found just inside exons near exon boundaries (the most common type of motif in this region). Very few motifs were found in 3′ UTR regions.

Figure 3. Longer introns have more predicted secondary structure.

a) The density of hits (the number of RNAz hits with RNAz score >0.5 divided by the total number of windows searched) is plotted against the length of the intron. You can see that longer introns have a higher density of RNAz hits. b) The density of predicted paired bases also increases with the length of the intron. c) The density of predicted paired bases is plotted as a function of the relative position within the intron, for four different length groups of introns. You can see that longer introns (light blue and yellow curves) have a higher density of predicted paired bases across their entire length than shorter introns (the dark blue and pink curves).

Figure 4. Predicted base pairs are preferentially found just inside exon boundaries.

Locations of predicted base pairs were tabulated separately for four length categories of motifs (dark blue = 0–100 base long motifs, pink = 100–200 bases, yellow = 200–300 bases, light blue = 300–400 bases). These locations of predicted base pairing are plotted near the a) start codon; b) stop codon; c) 5′ splice site; and d) 3′ splice site. Predicted base-pairs involved in secondary structure are most common just inside exon boundaries, and many of these base-pairs are contained in short predicted secondary structures (0–100 bp).

Figure 5. The pattern of sequence conservation near exon boundaries cannot explain the secondary structure peak just inside exon boundaries.

The relative position within the exon is plotted versus the fraction of predicted base-pairs and sequence conservation for a) 5′-most exons; b) internal exons; and c) 3′-most exons. The peak in predicted secondary structure inside the exon boundary is present regardless of whether sequence conservation rises or drops near the exon boundary.

Figure 6. Density of predicted exonic secondary structure increases with the number of introns.

The density of predicted paired bases within exons increases with the total number of introns in the gene.