Protein-coding structured RNAs: A computational survey of conserved RNA secondary structures overlapping coding regions in drosophilids

Abstract

Functional RNA elements can be embedded also within exonic sequences coding for functional proteins. While not uncommon in viruses, only a few examples of this type have been described in some detail for eukaryotic genomes. Here we use RNAz and RNAcode, two comparative genomics methods that measure signatures of stabilizing selection acting on RNA secondary structure and peptide sequence, resp., to survey the fruit fly genomes. We estimate that there might be on the order of 1000 loci that are subject to dual selection pressure. The used genome-wide screens also expose the limitations of the currently available methods.

Introduction

The worlds of protein-coding genes and those of non-coding RNAs (ncRNAs) are often seen as clearly separated. A small number of examples from both prokaryotes and eukaryotes, however, demonstrates that this is not strictly true, see [1] for a recent review. The best studied case in animals is the Steroid receptor activator gene (SRA). Originally characterized as a non-coding RNA with a distinctive secondary structure [2], it was later found to have isoforms coding for the functional protein SRAP, see [3]. SRA is probably the most extreme example, as nearly the complete transcript is covered by both conserved RNA structure and protein-coding region. At the other extreme, however, structured RNA motifs, such as selenocystein insertion elements (SECIS), internal ribosome entry sites (IRES), or mRNA localization signals are not at all frequent in UTRs of protein-coding transcripts [4]. In some cases, in particular in viruses, such structured elements are found in coding regions. The software tool RNAdecoder [5], which implements a comparative method for finding and folding RNA secondary structures within protein-coding regions, provided statistical evidence for frequent superpositions of RNA structure and coding sequences [6]. Nevertheless, systematic genome-wide analyses of this phenomenon have not been published to date.

The overwhelming majority of annotated coding regions translates to proteins with more than 30 amino acids. The discovery of the very short, independently encoded Tarsal-less peptides [7], [8], however, suggests that more small ORFs of this type might be hidden in the genomic DNA. Examples such as plant ENOD40 [9] with its short ORFs embedded in a heavily structured RNA, furthermore, hint at a possible association of functional secondary structure with coding capacity in such atypical transcripts. These, however, are likely to have escaped standard gene annotation procedures.

We therefore start our survey of the drosophilid genomes with the independent prediction of evolutionarily conserved RNA secondary structures and of open reading frames with evidence of stabilizing selection acting on the peptide sequence. To this end we employ RNAz [10], [11] and RNAcode [12], respectively.