Ribozymes present the most primitive catalytic systems in Biology, and might have played a central role in jumpstarting life. A major class of ribozymes, referred to as small endonucleolytic ribozymes catalyze site-specific RNA cleavage. While the structural and mechanistic features of these RNAs have been studied extensively, we have little understanding about their evolutionary histories. Enzymes acquire novel functions through adaptive walks along paths virtually neutral to mutations. This involves gradual acquisition of new functions while conserving its existing ones till the emergence of an ‘optimally evolved’ catalyst through intermediate sequences exhibiting both functions. We explored the fitness landscapes of two functional RNAs – the VS and hairpin endonucleolytic ribozymes, by rational design, in-silico structure prediction and in-vitro cleavage assays and discovered multiple intersecting neutral networks that link the two distinct functions. Certain intersections represent sequences that could potentially adopt structures corresponding to both VS and hairpin folds and are catalytically active toward both VS and hairpin substrates. Catalytic promiscuity in primitive RNA might have driven exploration of sequence space and eventually certain sequences could’ve acquired a new function on encountering a novel functional space. Intersection of neutral networks therefore provide a plausible evolutionary mechanism for the acquisition of new function in catalytic RNA. Subsequently, the gene for intersection sequences could duplicate and diverge by adaptive evolution to more optimal activities relieving constraints of dual and therefore decreased activities. These optimal sequences could use more sophisticated catalytic strategies and have more robust folding properties exemplified by wild-type VS and hairpin ribozymes. This work paves a way for further investigations into the neutral networks of other related ribozymes and might provide insights into their evolutionary origins, enriching our understanding of RNA evolution in general, and in the context of primitive life prevalent in the RNA World.

Emergence of ribonuclease activity in ribozymes through intersection of neutral networks

255th National Meeting (2018)

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