Trends in Genetics
Genome AnalysisRapid evolution of noncoding RNAs: lack of conservation does not mean lack of function
Introduction
Recent transcriptome analyses have shown that most of the metazoan genome is transcribed 1, 2. Using cDNA cloning 3, 4, 5, 6, 7, 8, 9 and genome tiling arrays 10, 11, 12, 13, 14, 15, 16, researchers have detected surprisingly large numbers of candidate non protein-coding RNAs (ncRNAs), many of which appear to be localized to the nucleus and/or lack polyadenylation [15]. The function – if any – of these transcripts remains a matter of debate 17, 18, the resolution of which will have important implications for our understanding of genome biology.
Wang and colleagues recently reported that non-protein-coding transcripts identified in mouse cDNA collections are poorly conserved and therefore argued that they are likely to be non-functional [19]. By contrast, many members of known functional classes of ncRNAs – including microRNAs (miRNAs) and small nucleolar RNAs (snoRNAs) – are well-conserved across a diverse range of species 4, 5, 6, 7. Candidate ncRNAs however average ∼2 kb in length 9, 20 and hence are different from miRNAs and snoRNAs (which are typically 21–25 nt and 60–300 nt, respectively), and it might be more meaningful to compare their evolution with longer ncRNAs whose functions have been validated. Well-studied large ncRNAs such as Xist and Air are poorly conserved, although there are small segments of their sequences that are highly conserved 21, 22, 23, 24, 25. Therefore, the possibility arises that different types of ncRNAs can exhibit different patterns of sequence conservation. Here we provide evidence that supports this hypothesis and discuss the possible interpretations and implications of our results.
Section snippets
Analysis of functional ncRNA conservation
There are many types of functional ncRNAs: transfer RNAs (tRNAs), ribosomal RNAs (rRNAs) and small nuclear (spliceosomal) RNAs have been known for many years and have core functions in RNA processing and translation. However, in recent years, it has become evident that there are increasing numbers of other ncRNAs that regulate gene expression in different ways in complex organisms, including miRNAs, snoRNAs and other miscellaneous ncRNAs (see Ref. [26] and references therein). These regulatory
Known miRNAs and snoRNAs are well-conserved but other ncRNAs are not
Most of the known miRNAs are highly conserved, with >90% sequence identity between human and mouse (Figure 1a). This level of purifying selection is even greater than that observed for mRNAs or even the protein-coding sequences within mRNAs (Figure 1c,i), which in turn are more highly conserved than introns (Figure 1f). Strong miRNA conservation is hardly surprising given that phylogenetic conservation has been a criterion used to identify miRNAs [31].
Most snoRNAs in mammals have been
Interpretations and implications
We have provided evidence that different classes of functional ncRNAs are evolving differently, compared with one another and with protein-coding sequences. Why should this be, and what is its significance?
The known miRNAs and snoRNAs are well conserved, and there are several probable reasons for this conservation. First, both function by hybridization to other nucleic acids. For miRNAs, which are short, a consequence is that even slight changes in sequence can fundamentally alter function.
Concluding remarks
Our findings have implications for the criteria used to assess the significance of candidate ncRNAs. As noted earlier, others have taken the lack of primary sequence conservation to mean that thousands of putative mouse ncRNAs are non-functional [19]. The evidence presented here indicates that this conclusion might be premature, and that alternative criteria to assess the function of longer ncRNA candidates are needed. It has been suggested that folding energy predictions can be used to
Update
Recently, Willingham et al. [46] used sequence homology to identify 500 human orthologs of mouse ncRNAs identified by RIKEN. They designed siRNAs for functional screens and using cell-based reporter assays, identified a new ncRNA repressor of the nuclear transcription factor of activated T cells (NFAT), in addition to others that appear to be either essential for cell viability or involved in hedgehog signaling 46, 47. The identification of patches of conserved sequences between ncRNAs can be
Acknowledgements
This work was supported by research grants from the Australian Research Council to J.S.M. M.C.F. is a University of Queensland Postdoctoral Fellow. K.C.P. is supported by an NHMRC Medical Postgraduate Research Scholarship. We thank Constance Cepko and Tracy Young for providing us with information on TUG1 ncRNAs; Melanie Ginger and Jeff Rosen for providing a pre-print of their paper on PINC/GB7 ncRNA; and Albin Sandelin for assistance with Figure 1. We also thank the editor and two anonymous
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These authors contributed equally to this work.