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Evolution of Helix Formation in the Ribosomal Internal Transcribed Spacer 2 (ITS2) and Its Significance for RNA Secondary Structures

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Abstract

Helices are the most common elements of RNA secondary structure. Despite intensive investigations of various types of RNAs, the evolutionary history of the formation of new helices (novel helical structures) remains largely elusive. Here, by studying the nuclear ribosomal Internal Transcribed Spacer 2 (ITS2), a fast-evolving part of the eukaryotic nuclear ribosomal operon, we identify two possible types of helix formation: one type is “dichotomous helix formation”—transition from one large helix to two smaller helices by invagination of the apical part of a helix, which significantly changes the shape of the original secondary structure but does not increase its complexity (i.e., the total length of the RNA). An alternative type is “lateral helix formation”—origin of an extra helical region by the extension of a bulge loop or a spacer in a multi-helix loop of the original helix, which does not disrupt the pre-existing structure but increases RNA size. Moreover, we present examples from the RNA sequence literature indicating that both types of helix formation may have implications for RNA evolution beyond ITS2.

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Acknowledgments

The authors thank Michael Zuker (Institute for RNA Science and Technology, SUNY Albany, USA) for helpful comments on two different conformations of Helix 1 and for preparing probability dot plots on Pseudomuriella aurantiaca. We also acknowledge two anonymous reviewers for their valuable comments on the manuscript. This study was supported by the University of Cologne and the Heinrich-Hertz-Stiftung (Ministerium für Innovation, Wissenschaft, Forschung und Technologie des Landes Nordrhein-Westfalen, Germany; fellowship to LC).

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  1. Universität zu Köln, Biozentrum Köln, Botanisches Institut, Zülpicher Str. 47b, 50674, Köln, Germany

    Lenka Caisová & Michael Melkonian

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  1. Lenka Caisová
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  2. Michael Melkonian
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Corresponding author

Correspondence to Lenka Caisová.

Electronic supplementary material

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ESM 1

ITS2 alignment of the 112 Desmodesmus taxa. The secondary structure of ITS2 is described by a bracket notation, note that the alignment of Helix 1 is based on the dichotomous conformation, whereas the alignment of Helix 3 is derived from the non-dichotomous conformation. The alignment contains information on the 3′ end of the 5.8S rRNA (C3/E processing site) and the 5′ end of the 28S rRNA (C1 processing site) according to Côté et al. (2002). It also includes the universal numbering system (Caisová et al. 2011, 2013), and a mask (Mask_Phylogeny) indicating positions used for the phylogenetic analyses

ESM 2

ITS2 alignment of the 12 Pseudomuriella taxa. The secondary structure of ITS2 is described by a bracket notation. The alignment contains information on the 3′ end of the 5.8S rRNA (C3/E processing site) and the 5′ end of the 28S rRNA (C1 processing site) according to Côté et al. (2002). It also includes the universal numbering system (Caisová et al. 2011, 2013). Note that Helix 1 of P. cubensis KF2 cannot be unambiguously aligned with all other strains examined because of its unique sequence

ESM 3

Evolution of dichotomous helix formation of Helix 1 within the Sphaeropleales. Secondary structure diagrams of Helix 1 of the Sphaeropleales and its relatives (Chaetophorales, Chaetopeltidales and Oedogoniales—CCO group) were plotted against a simplified phylogram derived from supplementary material Fig. S3 in Caisová et al. (2013). The CCO group and three sequences of the Sphaeropleales (=Sphaeropleaceae) possess only non-dichotomous version of Helix 1, whereas the remaining organisms (the Sphaeropleales with exclusion of the Sphaeropleaceae, Pseudomuriella and Desmodesmus) display the dichotomous pattern of Helix 1. The intermediate step between the non-dichotomous (=ancestral) and dichotomous (=derived) pattern of Helix 1 is represented by the genus Pseudomuriella, namely by P. aurantiaca in which two thermodynamically indistinguishable structures of Helix 1 exist. One of these is a single (non-dichotomous) helix while the other is the dichotomous form. The P. engadinensis sequences differ in several positions such that now the dichotomous form is thermodynamically more likely. A sequence of transitions leading from the dichotomous (and non-dichotomous) to the non-dichotomous (=ancestral) pattern of Helix 1 in Pseudomuriella is depicted. (Note that only Pseudomuriella sequences with the same number of nucleotides in Helix 1 are shown). Substitutions leading to the ancestral (=non-dichotomous) pattern of Helix 1 in Pseudomuriella are indicated by red and blue colors and by arrows in the appropriate color code. The block of homologous basepairs identified in the variable part of Helix 1 between Sphaeropleaceae and Pseudomuriella is shown in blue. Note that the last nucleotide at the 3′ end of this block (blue arrow) underwent a substitution from A (Sphaeropleaceae) → U (Pseudomuriella). The corresponding regions in both the non-dichotomous and the dichotomous pattern in Pseudomuriella were highlighted using a grey line. Energy values are shown for the whole ITS2 module and refer to a total Gibbs energy (dG—kcal/mol). The 5′ and 3′ termini are labeled. The most parsimonious origin of the dichotomous pattern of Helix 1 is specified by a yellow star. The corresponding regions in all schemes in the CCO group and the Sphaeropleales were highlighted using black and grey colors. (Note that the black color refers to the region from universal basepair 4/15 to universal basepair 9/10. Branches marked in bold were supported by RAxML/MP ≥70 % bootstrap and Bayesian posterior probability (MrBayes) = 1.00. Nine interrupted branches (//) have been shortened to 25, 30, 50, 70 or 75 % of their original length

ESM 4

Dichotomous helix formation in an existing Helix 3. Secondary structure diagrams of two closely related species of green algae: a Desmodesmus arthrodesmiformis DQ417537 and b Desmodesmus armatus AM410661, showing nucleotide rearrangements at the origin of dichotomous helix formation. The brown outlines delineate homologous regions between the non-dichotomous (a-I, b-I) and dichotomous (a-II, b-II) structures of both organisms. The substitutions leading to a different positioning of dichotomous helix formation (a-II vs b-II) are shown in red. The numbering system was adopted from Caisová et al. (2011, 2013). Energy values are shown for the whole ITS2 module and refer to a total Gibbs energy (dG—kcal/mol). Note that the energy values for b-I diagram vary depending on the dichotomous or non-dichotomous conformation of Helix 1. The 5′ and 3′ termini are labeled

ESM 5

Phylogeny of 112 Desmodesmus taxa using ITS2 sequence comparison. The tree topology was reconstructed using RAxML, and was based on 198 aligned ITS2 characters. The branch separating Desmodesmus clade 2 from the remaining Desmodesmus sequences was used for rooting the tree. The support at branches (from left to right) refers to bootstrap values of the RAxML/NJ/MP analyses and to Bayesian posterior probabilities (MrBayes). One interrupted branch (//) has been shortened to 50 % of its original length. Two taxa investigated in Online Resource 4 are highlighted in red. Taxa having two different conformations (dichotomous and non-dichotomous) of Helix 1 and/or Helix 3 are highlighted with colour ranges (grey, blue, green). Note that taxa without any background display only one conformation (dichotomous) of Helix 1 and only one conformation (non-dichotomous) of Helix 3, representing typical helical conformations of the vast majority of taxa/sequences in the Sphaeropleales

ESM 6

Melting probability dot plots of Helix 1 in Pseudomuriella aurantiaca. The sequence of Helix 1 was folded at temperatures ranging from 1.5 to 83.5 °C. a Schematic presentation of temperature-induced changes in structural conformation of Helix 1 in two strains of Pseudomuriella aurantiaca CCAP 249/1 and KF 43. At lower temperatures (<32 °C) the dichotomous structure is the only fold predicted. At 32 °C, the non-dichotomous structure appears and the probability of this structure increases with temperature (for details see b). At about 52 °C both structures are equally likely and only at temperatures higher than 52 °C the non-dichotomous structure dominates. b 165 dot plot graphs showing the probability of dichotomous and non-dichotomous structures of Helix 1 in P. aurantiaca. The header in the images contains the same name (Probability Dotplot for Pseudomuriella_aurantiaca) and the temperature (as for example 32, which means 32 °C; 32_5 means 32.5 °C.)

ESM 7

Different scenarios of the hypothetical origin of the ITS2 module. Schematic presentations of three main possible scenarios (and their modifications) of the origin of the ITS2 module are outlined. a Only dichotomous helix formation is employed. Scheme 1—B9 Helix in Bacteria and Archaea. Scheme 2—prolongation of the apical part (variable region) of the B9 Helix in the putative ancient eukaryote. Schemes 3, 4—invagination of the apical part of the B9 Helix (yellow arrow) and origin of Helix 2 and 3 (H2, H3) by dichotomous helix formation. Scheme 5—prolongation of Helix 2, 3 and invagination of their apical parts (yellow arrows) resulting in the origin of Helix 4 (H4). Scheme 6—a typical ITS2 module with four helices (H1–H4) that are interconnected with the multi (5)-helix loop. b Only lateral helix formation is employed. Scheme 1 is identical to schematic 1 in a. Scheme 2—prolongation of the B9 Helix and expansion of its apical loop, scheme 3—origin of helices 1–4 (H1–H4) using lateral helix formation. c Combination of dichotomous and lateral helix formation employed. There are three different scenarios possible: one is outlined as a schematic drawing and the other two are described in Figure. In the drawing, the first four schemes (1–4) are identical to schemes 1–4 in a. Schemes 5, 6 refer to the origin of Helix 1 (H1) using lateral helix formation and schemes 7, 8 show the origin of Helix 4 (H4) again using lateral helix formation. For the most likely scenario see Results, Fig. 4 and Online Resource 8

ESM 8

Animation of the most likely scenario on the hypothetical evolution of the ITS2 module in eukaryotes. Details about the hypothetical origin of ITS2 are given in Discussion. To run this movie, click the Slide Show button

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Caisová, L., Melkonian, M. Evolution of Helix Formation in the Ribosomal Internal Transcribed Spacer 2 (ITS2) and Its Significance for RNA Secondary Structures. J Mol Evol 78, 324–337 (2014). https://doi.org/10.1007/s00239-014-9625-0

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