The ribosome is the cell's protein-building workbench and ribonucleic acids, the molecules we call RNA, are key tools perform a host of vital functions in cells.According to a new analysis, even before the ribosome's many working parts were recruited for protein synthesis, proteins also were on the scene and interacting with RNA. This finding challenges a hypothesis about the early evolution of life.

The "RNA world" hypothesis first appeared in 1986 and posits that the first stages of molecular evolution involved RNA and not proteins, and that proteins (and DNA) emerged later, said University of Illinois crop sciences and Institute for Genomic Biology professor Gustavo Caetano-Anollés, who led a new study. "I'm convinced that the RNA world (hypothesis) is not correct. That world of nucleic acids could not have existed if not tethered to proteins."

The ribosome is a "ribonucleoprotein machine," a complex that can have as many as 80 proteins interacting with multiple RNA molecules, so it makes sense that this assemblage is the result of a long and complicated process of gradual co-evolution, said Caetano-Anollés. Furthermore, "you can't get RNA to perform the molecular function of protein synthesis that is necessary for the cell by itself."

The RNA world hypothesis makes some assumptions about the evolutionary origins of the ribosome, Caetano-Anollés said. The most fundamental of these assumptions is that the part of the ribosome that is responsible for protein synthesis, the peptidyl transferase center (PTC) active site, is the most ancient.

In the new analysis, Caetano-Anollés and graduate student Ajith Harish subjected the universal protein and RNA components of the ribosome to rigorous molecular analyses – mining them for evolutionary information embedded in their structures. They also analyzed the thermodynamic properties of the ribosomal RNAs and used this information to generate timelines of the evolutionary history of the ribosomal RNAs and proteins.

These two, independently generated "family trees" of ribosomal proteins and ribosomal RNAs showed "great congruence" with one another, Caetano-Anollés said. Proteins surrounding the PTC, for example, were as old as the ribosomal RNAs that form that site. In fact, the PTC appeared in evolution just after the two primary subunits that make up the ribosome came together, with RNA bridges forming between them to stabilize the association.

The timelines suggest to 
Caetano-Anollés that the PTC appeared well after other regions of the protein-RNA complex, Caetano-Anollés said. He says that further suggests that proteins were around before ribosomal RNAs were recruited to help build them, and second, that the ribosomal RNAs were engaged in some other task before they picked up the role of aiding in protein synthesis, he said.

"This is the crucial piece of the puzzle," Caetano-Anollés said. "If the evolutionary build-up of ribosomal proteins and RNA and the interactions between them occurred gradually, step-by-step, the origin of the ribosome cannot be the product of an RNA world. Instead, it must be the product of a ribonucleoprotein world, an ancient world that resembles our own. It appears the basic building blocks of the machinery of the cell have always been the same from the beginning of life to the present: evolving and interacting proteins and RNA molecules."

A chronological representation of the evolution of the ribosome shows that very early in ribosomal evolution (nd<0.3) rRNA helices interacted with r-proteins to form a processivity core that mediated nucleotide interactions, which later (nd = 0.3) served as center for coordinated and balanced RNP accretion leading to modern ribosomal function. The purple structure indicates extant mRNA, which is used as structural reference for location of primitive functional centers. We envision the primordial ribosome had replicative functions that likely involved RNA, so the mRNA molecule from the crystallographic model should be regarded as placeholder for the ancient coding molecule. rRNA is rendered as ribbon representation, mRNA and proteins as rendered as space-filling representations. doi:info:doi/10.1371/journal.pone.0032776.g007

University of California at San Diego research professor Russell Doolittle, who was not involved in the study, remains puzzled,  by "the notion that some early proteins were made before the evolution of the ribosome as a protein-manufacturing system." He wondered how – if proteins were more ancient than the ribosomal machinery that today produces most of them –"the amino acid sequences of those early proteins were 'remembered' and incorporated into the new system."

Caetano-Anollés agreed that this is "a central, foundational question" that must be answered.

"It requires understanding the boundaries of emergent biological functions during the very early stages of protein evolution," he said. However, he said, "the proteins that catalyze non-ribosomal protein synthesis – a complex and apparently universal assembly-line process of the cell that does not involve RNA molecules and can still retain high levels of specificity – are more ancient than ribosomal proteins. It is therefore likely that the ribosomes were not the first biological machines to synthesize proteins."

Caetano-Anollés also noted that the specificity of the ribosomal system "depends on the supply of amino acids appropriately tagged with RNA for faithful translation of the genetic code. This tagging is solely based on proteins, not RNAs," he said. This suggests, he said, that the RNA molecules began as co-factors that aided in protein synthesis and fine-tuned it, resulting in the elaborate machinery of the ribosome that exists today.

Citation: Harish A, Caetano-Anollés G (2012) Ribosomal History Reveals Origins of Modern Protein Synthesis. PLoS ONE 7(3): e32776. doi:10.1371/journal.pone.0032776