Comparative genomics beyond sequence-based alignments: RNA structures in the ENCODE regions

Abstract

Recent computational scans for non-coding RNAs (ncRNAs) in multiple organisms have relied on existing multiple sequence alignments. However, as sequence similarity drops, a key signal of RNA structure-frequent compensating base changes-is increasingly likely to cause sequence-based alignment methods to misalign, or even refuse to align, homologous ncRNAs, consequently obscuring that structural signal. We have used CMfinder, a structure-oriented local alignment tool, to search the ENCODE regions of vertebrate multiple alignments. In agreement with other studies, we find a large number of potential RNA structures in the ENCODE regions. We report 6587 candidate regions with an estimated false-positive rate of 50%. More intriguingly, many of these candidates may be better represented by alignments taking the RNA secondary structure into account than those based on primary sequence alone, often quite dramatically. For example, approximately one-quarter of our predicted motifs show revisions in >50% of their aligned positions. Furthermore, our results are strongly complementary to those discovered by sequence-alignment-based approaches-84% of our candidates are not covered by Washietl et al., increasing the number of ncRNA candidates in the ENCODE region by 32%. In a group of 11 ncRNA candidates that were tested by RT-PCR, 10 were confirmed to be present as RNA transcripts in human tissue, and most show evidence of significant differential expression across tissues. Our results broadly suggest caution in any analysis relying on multiple sequence alignments in less well-conserved regions, clearly support growing appreciation for the biological significance of ncRNAs, and strongly support the argument for considering RNA structure directly in any searches for these elements.

The tedious task of finding homologous noncoding RNA genes

“…problems due to structural variation over large phylogenetic distances, many novel structured RNA candidates can be inferred from covarying patterns in structurally conserved RNAs, as demonstrated, for example, on the ENCODE data (Washietl et al 2007;Torarinsson et al 2008). Once a novel ncRNA has been identified by one of these approaches, however, we are back to the problems of homology-based methods to identify additional family members.…”

LocARNA-P: Accurate boundary prediction and improved detection of structural RNAs

“…The idea of revising the alignment for ncRNA prediction, pioneered by MSARi (Coventry et al 2004), is also realized in the EM-based approach CMfinder (Yao et al 2006), which extends the idea from local sequence motif finders such as MEME to the problem of finding local RNA structure motifs. Due to its high computational demands, CMfinder has never been applied to whole eukaryotic genomes; for example, in (Torarinsson et al 2008), it has been applied to the ENCODE region, covering only 1% of the human genome.…”

Improved RNA secondary structure prediction by maximizing expected pair accuracy

“…The multiple functions of ncRNA have fundamentally changed the original formulation of the Central Dogma of Biology, where it was originally thought that proteins were the only final effectors of genetics. Both the discovery (Washietl et al 2005;Uzilov et al 2006;Torarinsson et al 2008) and the analysis of function of ncRNA depend on the determination of structure.…”