Experimental Resurrection of Ancestral Mammalian CPEB3 Ribozymes Reveals Deep Functional Conservation

Abstract

Self-cleaving ribozymes are genetic elements found in all domains of life, but their evolution remains poorly understood. A ribozyme located in the second intron of the cytoplasmic polyadenylation binding protein 3 gene (CPEB3) shows high sequence conservation in mammals, but little is known about the functional conservation of self-cleaving ribozyme activity across the mammalian tree of life or during the course of mammalian evolution. Here, we use a phylogenetic approach to design a mutational library and a deep sequencing assay to evaluate the in vitro self-cleavage activity of numerous extant and resurrected CPEB3 ribozymes that span over 100 My of mammalian evolution. We found that the predicted sequence at the divergence of placentals and marsupials is highly active, and this activity has been conserved in most lineages. A reduction in ribozyme activity appears to have occurred multiple different times throughout the mammalian tree of life. The in vitro activity data allow an evaluation of the predicted mutational pathways leading to extant ribozyme as well as the mutational landscape surrounding these ribozymes. The results demonstrate that in addition to sequence conservation, the self-cleavage activity of the CPEB3 ribozyme has persisted over millions of years of mammalian evolution.

Keywords: CPEB3; RNA; ancestral sequence resurrection; fitness landscape; phylogenetics.

Fig. 1.

Sequence conservation and secondary structure of CPEB3 ribozyme. (a) Mapping and conservation of the CPEB3 ribozyme. Protein, mRNA and gene are adapted from Salehi-Ashtiani et al. (2006). Four notable domains are identified in the protein primary structure (Q, glutamine-rich domain; RRM, RNA-binding domains; Znf, zinc finger). Vertical dividers in the mRNA indicate splice sites. Tissue-specific untranslated exons are marked below the gene with letters (L, liver; T, testis; B, brain). Translated exons are indicated as large vertical lines in the “gene” diagram. Self-cleaving CPEB3 ribozyme location is indicated as Rz in the second intron and the human CPEB3 sequence is shown expanded below with the cleavage site indicated by a gray arrowhead. An asterisk marks the human SNP (U36C). Plots indicate the conservation of each nucleotide in the consensus sequence of the 100 identified mammalian CPEB3 sequences within each clade. (b) Secondary structure of the consensus ribozyme sequence. Triangle indicates self-cleavage site and black circles indicate cleaved sequence. Nucleotides are colored according to conservation across the Class Mammalia. Asterisk indicates the location of a SNP (U36C) in the human ribozyme sequence.

Fig. 2.

In vitro activity of extant and ancestral mammalian CPEB3 ribozymes mapped onto the mammalian tree of life. Phylogenetic tree derived from the 99 mammalian species with identified CPEB3 ribozyme sequences. Each node indicates a ribozyme sequence that is either found in an extant species (outer) or represents a predicted ancestral sequence (inner). The color of the node indicates the in vitro self-cleaving ribozyme activity (see inset). Animal silhouettes are colored according to their respective ribozyme activities. Square nodes indicate a single highly functional, highly conserved ancestral sequence. Circle nodes indicate ribozyme sequences that were biochemically assessed using high-throughput sequencing. Circle nodes with asterisks indicate sequences assessed using gel electrophoresis. Triangle nodes indicate a sequence with predicted ribozyme activity based on mutational effects observed in the data. Red “x” indicates a mutation at position G1. Species names are indicated in supplementary figure S1, Supplementary Material online.

Fig. 3.

Distributions of ribozyme activities. (a) Distributions of ribozyme activities for taxonomic groups. Dashed lines indicate mean of ribozyme activities. Plots are colored according to the median ribozyme activity. Extant sequences with predicted low ribozyme activities were assumed to have ∼0.05 activity for the distribution. (b) Ribozyme activity of all 27,648 sequences in the phylogenetic mutational library plotted as a function of mutations from the highly conserved ancestral sequence. Each node indicates a unique ribozyme sequence and the color and size of the node indicate ribozyme activity. The number of sequences (n) that correspond to each mutational distance from the ancestral sequence is shown. The dashed line passes through the mean activity at each mutational distance.

Fig. 4.

Genotype network and mutational neighborhoods. (a) A “top down” view of the CPEB3 ribozyme genotype network as a force-directed graph. Nodes represent individual sequences. Ribozyme activity is represented with node color and size (see inset for color scale). Only nodes with ribozyme activity > 0.5 are shown. Edges connect nodes that differ by a single nucleotide change and are colored as the average of the two connected nodes. Clusters of nodes that are outlined in black all share a common group of mutations, which are labeled. Extant ribozyme sequences are labeled: 1 = opossum/koala, 2 = Tasmanian devil/common wombat/wallaby, 3 = bushtail possum, 4 = rock hyrax, 5 = mouse and several other rodents, 6 = pika/marmot/and several other rodents, 7 = chinchilla, A = ancestral sequence, H = human ribozyme. (b) The mutational neighborhood surrounding specific ribozyme sequences. The center node represents the ribozyme variant labeled above. Each concentric circle represents all the sequences at that mutational distance (1–4) that are accessible to the central ribozyme sequence through mutational pathways while maintaining ribozyme activity > 0.05. Edges are colored based on the ribozyme activity of the genotype with higher mutations (see color bar).

Fig. 5.

Potential evolutionary pathways from the ancestral sequence to extant primates and marsupials. (a) The top panel depicts the mutational pathways from the ancestral sequence to 29 extant primate sequences. The bottom panel depicts the mutational pathways between the ancestral sequence and six extant marsupial sequences. Each data point (node) indicates the relative ribozyme activity (y-axis) and the number of mutations from the ancestral sequence (x-axis). Nodes have been horizontally jittered for help with visualization. Color of nodes indicates ribozyme activity (scaled as in other figures). Nodes of ancestral and extant sequences have black edges and are labeled with species names and specific mutations. Nodes without black edges are predicted intermediate sequences. Gray lines (edges) connect nodes that differ by a single mutation. (b) The top secondary structure indicates the mutations found in primates relative to the ancestral sequence. The bottom structure shows mutations found in marsupials. Mutational nucleotides are colored according to the single mutation effect on the ribozyme activity of the ancestral sequence, with color scaled as identical to other figures. The triangle indicates the self-cleavage site and the upstream sequence is indicated as black dots.