Robust Identification of Noncoding RNA from Transcriptomes Requires Phylogenetically-Informed Sampling

Abstract

Noncoding RNAs are integral to a wide range of biological processes, including translation, gene regulation, host-pathogen interactions and environmental sensing. While genomics is now a mature field, our capacity to identify noncoding RNA elements in bacterial and archaeal genomes is hampered by the difficulty of de novo identification. The emergence of new technologies for characterizing transcriptome outputs, notably RNA-seq, are improving noncoding RNA identification and expression quantification. However, a major challenge is to robustly distinguish functional outputs from transcriptional noise. To establish whether annotation of existing transcriptome data has effectively captured all functional outputs, we analysed over 400 publicly available RNA-seq datasets spanning 37 different Archaea and Bacteria. Using comparative tools, we identify close to a thousand highly-expressed candidate noncoding RNAs. However, our analyses reveal that capacity to identify noncoding RNA outputs is strongly dependent on phylogenetic sampling. Surprisingly, and in stark contrast to protein-coding genes, the phylogenetic window for effective use of comparative methods is perversely narrow: aggregating public datasets only produced one phylogenetic cluster where these tools could be used to robustly separate unannotated noncoding RNAs from a null hypothesis of transcriptional noise. Our results show that for the full potential of transcriptomics data to be realized, a change in experimental design is paramount: effective transcriptomics requires phylogeny-aware sampling.

Small Regulatory RNAs of Rickettsia conorii

“…Despite the prevalence of sRNAs in many bacteria, there exists only limited evolutionary conservation and both species- and strain-specific sRNA catalogues are now documented22. A search of 400 transcriptomic datasets belonging to 40 different strains of bacteria and archaea has revealed that the ‘Goldilocks Zone’ (where species are neither too close nor too distant phylogenetically) for non-coding RNAs is rather narrow, indicating independent evolution of lineage-specific post-transcriptional machinery23. As further confirmatory evidence supporting this notion, a search for orthologs of 2208 non-coding RNAs within 1156 bacterial genomes reported in Rfam (a collection of non-coding RNA families)24, including members of Rickettsiales , also reveals limited taxonomic distribution and suggests a low degree of evolutionary conservation in a majority of these ncRNA families25.…”

Origin, Evolution, and Loss of Bacterial Small RNAs

“…High degrees of both intra- and inter-species polymorphism yield low sequence similarity, especially as compared to protein-coding genes, and sRNAs on the whole are known to be poorly conserved across broad evolutionary distances (6). Comparing the contents of Rfam (7)—a database that houses sequence and structural information for known sRNA families—to Pfam (8), an analogous database for protein families, only 60% of RNA families were found to be conserved between species of the same taxonomic family, as opposed to 90% for proteins (9). In evolutionary analyses, where a clear picture of relationships between gene sequences through time is critical in order to draw meaningful conclusions, this presents the unique challenge of distinguishing between an sRNA’s absence in a given lineage and the possibility that the sequence or structure of the gene has simply diverged too drastically to accurately trace its ancestry.…”

The genetic robustness of RNA and protein from evolutionary, structural and functional perspectives

“…The discovery of functional non-coding RNAs (ncRNAs), like transfer RNA and catalytic RNAs, led to the proposal of an ancestral RNA world, where RNA both catalyzes the reactions of life and encodes genetic information (3) . Despite the importance of non-coding RNAs to cellular function, many RNAs exhibit low sequence conservation and are not as broadly distributed as their proteinaceous brethren (4) . In contrast, proteins make up the bulk of the biomolecular contents of the cell, and are required for the majority of critical cellular structures and functions.…”