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. 2022 Sep 2;12(9):1373.
doi: 10.3390/life12091373.

Rolling Circles as a Means of Encoding Genes in the RNA World

Felipe Rivera-Madrinan  1 , Katherine Di Iorio  1 , Paul G Higgs  1
Affiliations

Rolling Circles as a Means of Encoding Genes in the RNA World

Felipe Rivera-Madrinan et al. Life (Basel). .

Abstract

The rolling circle mechanism found in viroids and some RNA viruses is a likely way that replication could have begun in the RNA World. Here, we consider simulations of populations of protocells, each containing multiple copies of rolling circle RNAs that can replicate non-enzymatically. The mechanism requires the presence of short self-cleaving ribozymes such as hammerheads, which can cleave and re-circularize RNA strands. A rolling circle must encode a hammerhead and the complement of a hammerhead, so that both plus and minus strands can cleave. Thus, the minimal functional length is twice the length of the hammerhead sequence. Selection for speed of replication will tend to reduce circles to this minimum length. However, if sequence errors occur when copying the hammerhead sequence, this prevents cleavage at one point, but still allows cleavage on the next passage around the rolling circle. Thus, there is a natural doubling mechanism that creates strands that are multiple times the length of the minimal sequence. This can provide space for the origin of new genes with beneficial functions. We show that if a beneficial gene appears in this new space, the longer sequence with the beneficial function can be selected, even though it replicates more slowly. This provides a route for the evolution of longer circles encoding multiple genes.

Keywords: RNA world; computer simulation; error threshold; rolling circle.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Figures

Figure 1
Figure 1
Mechanism of non-enzymatic rolling circle replication. Blue and green strands are complimentary plus and minus strands, each of which contains a ribozyme unit AZ and its complement za. After one circuit around the template, a double strand is created. After another circuit, a tail is produced. When the AZ motif is exposed in the tail, cleavage occurs, creating a linear strand that can circularize and begin the cycle anew.
Figure 2
Figure 2
Mutations when copying minimal length circles give three kinds of strands with different behavior. A mutation in the za motif (either z*, *a, or **) gives a non-cleaving circle which produces a complementary strand that can never cleave. A mutation in the AZ motif creates a halving circle which goes on to produce circles of half its own length. A mutation in both motifs produces a circle of double the original length that can stably replicate.
Figure 3
Figure 3
Mean number of strands of each length per cell. Colours indicate strand type: reproductive strands (green), non-cleaving strands (magenta), halving strands (blue), and non-circular fragments (yellow). (A), mutation probability u = 0. (B), mutation probability u = 0.15. (C), mutation probability u = 0.3. (D), mutation probability u = 0.45. uindel = 0 for all graphs.
Figure 4
Figure 4
Mean number of strands per cell of the four types as a function of mutation probability u. Colours indicate reproductive strands (green), non-cleaving strands (magenta), halving strands (blue), and non-circular fragments (yellow). Strands of different lengths of each type are combined.
Figure 5
Figure 5
Mean number of strands of each length per cell, beginning from a single 8-mer per cell, including point mutations with probability u = 0.15 and indels with probability uindel = 0.015. Colours indicate reproductive strands (green), non-cleaving strands (magenta), halving strands (blue), and non-circular fragments (yellow). (A) Simulation steps elapsed T = 2 × 104 (B) Simulation steps elapsed T = 2 × 106.
Figure 6
Figure 6
Comparison of reproductive rates of cells containing 8-mers with a beneficial gene and cells containing minimal-length 4-mers. The reproduction rate of the 8-mer cells depends on the size of the beneficial effect, β, and the circularization probability of the hammerhead, fcirc. There is no mutation in this figure: u = 0 and uindel = 0. (A) shows lower values of β, where the reproduction rate of the 8-mer cells is comparable to that of the 4-mer cells, or less. (B) shows higher values of β, where the reproduction rate of the 8-mer cells is much higher than the 4-mer cells.
Figure 7
Figure 7
Graph showing number of cells in a 100 cell population divided into those with beneficial genes and no 4-mers, those with 4-mers and no beneficial gene, those with both, and those with neither. Time is in simulation hours. The first arrow shows the point at which the beneficial gene was added with β = 5. The second arrow shows the point at which the first cell appears containing beneficial 8-mers but no 4-mers. u = 0.15 and uindel = 0.015 in this graph.
Figure 8
Figure 8
Percentage of (2 × 104) runs that result in takeover by the beneficial gene in a population of 100 cells. u = 0.15 and uindel = 0 for all runs.
Figure 9
Figure 9
Steady state distribution of lengths beginning from 8-mer sequences YzaB***A, with uindel = 0.015 and u = 0.15. (A), β = 5. (B), β = 1. Colours indicate reproductive strands (green), non-cleaving strands (magenta), halving strands (blue), and non-circular fragments (yellow).
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