Functionality or transcriptional noise? Evidence for selection within long noncoding RNAs

Abstract

Long transcripts that do not encode protein have only rarely been the subject of experimental scrutiny. Presumably, this is owing to the current lack of evidence of their functionality, thereby leaving an impression that, instead, they represent "transcriptional noise." Here, we describe an analysis of 3122 long and full-length, noncoding RNAs ("macroRNAs") from the mouse, and compare their sequences and their promoters with orthologous sequence from human and from rat. We considered three independent signatures of purifying selection related to substitutions, sequence insertions and deletions, and splicing. We find that the evolution of the set of noncoding RNAs is not consistent with neutralist explanations. Rather, our results indicate that purifying selection has acted on the macroRNAs' promoters, primary sequence, and consensus splice site motifs. Promoters have experienced the greatest elimination of nucleotide substitutions, insertions, and deletions. The proportion of conserved sequence (4.1%-5.5%) in these macroRNAs is comparable to the density of exons within protein-coding transcripts (5.2%). These macroRNAs, taken together, thus possess the imprint of purifying selection, thereby indicating their functionality. Our findings should now provide an incentive for the experimental investigation of these macroRNAs' functions.

Figure 1.

Nucleotide substitution and transversion rates are suppressed within macroRNA transcripts. Panels show the cumulative distributions of substitution (A,B) and transversion rates (C,D) as measured on macroRNA transcripts (red curves), and the same rates measured on nearby nonoverlapping AR sequence of matched length (black curves). (A) Mouse–human substitution rates; (B) mouse–rat substitution rates; (C,D) mouse–human and mouse–rat transversion rates. All macroRNA rates are significantly different from, and lower than, the putatively neutral AR rates (Kolmogorov-Smirnov test, P < 10⁻¹⁵ for all panels). The suppression of transversion rates in macroRNAs compared to AR sequence demonstrates that these observations are not a consequence of a higher density of highly mutable CpG sites within ARs, since the associated mutations are mainly transitions rather than transversions.

Figure 2.

Density of indel-purified segments in macroRNA transcripts. Shown are the IPS densities within macroRNA transcripts (red line) for 10 G+C content bins (horizontal axis), and the expected density based on the intergenic distribution of IPSs (black line; the gray band indicates 95% confidence intervals obtained by randomization; see Methods for details). The IPS densities within macroRNAs exceed significantly the levels expected in G+C-matched intergenic sequence, indicating the past action of purifying selection.

Figure 3.

Strong conservation of macroRNA promoters. Panels show the cumulative distributions of substitution (A,B) and transversion rates (C,D), as measured on the core putative promoter regions of macroRNA transcripts (red curves; 0–400 bp upstream of transcription start site), and the same rates on nearby AR sequence of the same length (black curves). Mouse macroRNA putative core promoter regions exhibit significantly suppressed substitution and transversion rates compared to selectively neutral ARs (P < 10⁻¹⁵ for all panels). This is true both for mouse–human (A,C) and mouse–rat (B,D) comparisons.

Figure 4.

Density of indel-purified segments in macroRNA promoters. Shown are the IPS densities within 400-bp regions upstream of macroRNA transcripts (red line) for 10 G+C content bins (horizontal axis), and the expected density based on the intergenic distribution of IPSs (black line; the gray band indicates 95% confidence intervals). IPS densities are substantially and significantly higher within putative core promoter regions.