Evolving Together: Cassandra Retrotransposons Gradually Mirror Promoter Mutations of the 5S rRNA Genes

Abstract

The 5S rRNA genes are among the most conserved nucleotide sequences across all species. Similar to the 5S preservation we observe the occurrence of 5S-related nonautonomous retrotransposons, so-called Cassandras. Cassandras harbor highly conserved 5S rDNA-related sequences within their long terminal repeats, advantageously providing them with the 5S internal promoter. However, the dynamics of Cassandra retrotransposon evolution in the context of 5S rRNA gene sequence information and structural arrangement are still unclear, especially: (1) do we observe repeated or gradual domestication of the highly conserved 5S promoter by Cassandras and (2) do changes in 5S organization such as in the linked 35S-5S rDNA arrangements impact Cassandra evolution? Here, we show evidence for gradual co-evolution of Cassandra sequences with their corresponding 5S rDNAs. To follow the impact of 5S rDNA variability on Cassandra TEs, we investigate the Asteraceae family where highly variable 5S rDNAs, including 5S promoter shifts and both linked and separated 35S-5S rDNA arrangements have been reported. Cassandras within the Asteraceae mirror 5S rDNA promoter mutations of their host genome, likely as an adaptation to the host's specific 5S transcription factors and hence compensating for evolutionary changes in the 5S rDNA sequence. Changes in the 5S rDNA sequence and in Cassandras seem uncorrelated with linked/separated rDNA arrangements. We place all these observations into the context of angiosperm 5S rDNA-Cassandra evolution, discuss Cassandra's origin hypotheses (single or multiple) and Cassandra's possible impact on rDNA and plant genome organization, giving new insights into the interplay of ribosomal genes and transposable elements.

Keywords: 35S-5S linkage; 5S promoter; 5S rDNA; Asteraceae; Cassandra; concerted evolution; long terminal repeats; plant genomes; retrotransposons; ribosomal genes; sequence mimicry; transposable elements.

Fig. 1.

LTR comparison of 81 Cassandra retrotransposons. We performed an all-against-all dotplot analysis, where similarities between defined windows (k = 12, allowed mismatches n = 3) are marked as a dot. If sequences show larger regions of similarity, multiple dots form linear patterns. Sequences are shaded according to the longest common substring, meaning sequences with a high number of matching windows are shaded in dark gray and white/light gray for sequences with a small number of similar sequence units. Each and every sequence shows at least a small diagonal, which information-wise can be mainly limited to the conserved region Cassandra sequences share with the 5S rDNA and a short promoter-flanking region. Cassandra LTR sequences show increased similarity within one plant family but tend to accumulate mutations for more distal ones. Visualization of phylogenetic relationships are indicated by colored lines and resemble the current taxonomy proposed by The Angiosperm Phylogeny Group (2016). Placing the Asteraceae Cassandra retrotransposons into this framework of typical plant Cassandra lengths, they are on the smaller side, due to their short LTR and internal sequences with median length values of 270 and 82 bp, respectively, similarly to Cassandras from the Rosaceae, Amaranthaceae, and ferns.

Fig. 2.

Similarity region shared by the 5S rRNA gene and Cassandra retrotransposonsin the Asteraceae and other plants. Differences from the 5S gene are highlighted in colors (A = red, T = green, C = blue, G = yellow). Key sequence variation in the C-Box is highlighted in bold. The arrangement of the 5S rDNA in a linked (L) or separated (S) configuration is indicated (see also Fig. 3). The regions of high sequence divergence between the Box motifs are marked with an orange rectangle.

Fig. 3.

35S- 5S linkage of ribosomal RNA genes is present in several, but not all, Asteraceae species. The graphs represent the low coverage assembly of the rRNA genes and show gene order, arrangement, and organization. Complete circular graphs represent the typical rDNA monomer for each species. Linked ribosomal genes (L) show a single circular graph including the 5S rDNA gene (marked by arrows; five instances). Separated ribosomal RNA genes (S) show two graphs (10 instances). For Scalesia atractyloides (*), read quality strongly impaired the alignments and prevented full circular assemblies. Nevertheless, the rRNA genes were assembled completely, with several copies being separated by spacers. A separated arrangement can be concluded even from the imperfect assembly of S. atractyloides rDNAs. Five of the Asteraceae show the 35S-5S linkage being neither restricted to species with the promoter shift (see H. umbraculigerum and B. hawaiensis) nor affecting all species with the shifted promoter (see Chrysanthemum indicum). The line thickness is associated to the coverage depth of individual contigs; however, these are not comparable across the species shown. Sequence representation is not to scale.

Fig. 4.

Framework for understanding the interplay between Cassandra retrotransposons and the 5S rDNA in the Asteraceae: summary, integrative overview, and data-based limitations. The Asteraceae phylogeny in the first column is based on Mandel et al. (2019).

Fig. 5.

Cassandra origin and evolution within the plant kingdom. We suggest a Cassandra origin by obtaining 5S rDNA promoter (and flanking) regions due to spatial association, e.g. transposition, recombination or eccDNA integration through an LTR-retrotransposon (a). Based on our data we assume Cassandra originating (star) either as a single event in early vascular plants (b—left side; purple star) or multiple origins (b—right side; red/yellow stars) in the plant kingdom. In the single origin scenario one originator formed multiple variants (purple/pink circles). Each of the Cassandra families, we observe today (purple/pink rectangles), is a successor of one of these variants. For the multiple origin hypothesis, the scenario slightly changes with each family (red/yellow rectangles) being assignable to one of the newly emerged Cassandras (red/yellow stars) within the plant kingdom.