Introduction The introns-first theory was published just over a decade ago (Poole et al. 1998; Jeffares et al.

1998), and aimed to account for the origin of mRNA within an evolutionary framework for the origin of genetically encoded protein synthesis in the late stages of the RNA world (see Fig. 1 for a summary). There are three aspects to this hypothesis: that mRNA arose by co-option of expressed non-functional RNA, that the co-opted RNAs were interspersed between functional RNA genes, and that the core of the spliceosome, some extant genes, and their introns may be relics from this very early period. The last of these is directly testable in principle. Introns-first is thus part of a much wider analysis of the expectations of the continuity of RNA systems from the RNA and ribonucleoprotein (RNP) worlds to modern organisms. This more general RNAcontinuity theory (Fig. 2a, see Penny and Collins 2009) is that many classes of RNA in modern eukaryotes have existed since these earlier phases; they are in our terminology relics, though of course the associated proteins would only have arisen after encoded protein synthesis either introns first or early. Figure 2b shows the contrast between the RNA-continuity model and the more common idea that an early complexity of RNA control mechanisms from then RNP world was lost in prokaryotes (archaea and bacteria) and reinvented in eukaryotes. The last decade has seen a major expansion in our expanded the classes of RNA that are known and the questions that need to be addressed. An early focus was on ubiquitous RNAs with a processing function (e.g. rRNA, tRNA, snRNA, small nucleolar RNA [snoRNa], srpRNA, RNase P, and RNase MRP) and the exon/intron structure of eukaryote genes (see Gilbert 1987; CavalierSmith 2002; Rodrguez-Trelles et al. 2006; Di Giulio 2008a, b, and references therein), but the finding of the widespread and complex roles of RNA in eukaryote cells (including RNAi) has broadened the

discussion to the extent we now refer to the RNA infrastructure of the eukaryote cell (Collins and Penny 2009). In particular, a range of regulatory RNAs have been identified in the last decade and include the many classes of small RNA involved in RNAi, such as miRNA, siRNA and piRNA, as well as their role in epigenetics either introns first or early. Figure 2b shows the contrast between the RNA continuity model and the more common idea that an early complexity of RNA control mechanisms from then RNP world was lost in prokaryotes (archaea and bacteria) and reinvented in eukaryotes. The last decade has seen a major expansion in our knowledge of the roles of RNA in eukaryotes and has expanded the classes of RNA that are known and the questions that need to be addressed. An early focus was on ubiquitous RNAs with a processing function (e.g. rRNA, tRNA, snRNA, small nucleolar RNA [snoRNa], srpRNA, RNase P, and RNase MRP) and the exon/intron structure of eukaryote genes (see Gilbert 1987; Cavalier-Smith 2002; Rodrguez-Trelles et al. 2006; Di Giulio 2008a, b, and references therein), but the finding of the widespread and complex roles of RNA in eukaryote cells (including RNAi) has broadened the discussion to the extent we now refer to the RNA infrastructure of the eukaryote cell (Collins and Penny 2009). In particular, a range of regulatory RNAs have been identified in the last decade and include the many classes of small RNA involved in RNAi, such as miRNA, siRNA and piRNA, as well as their role in epigenetics (Carthew and Sontheimer 2009). (Carthew and Sontheimer 2009)