- There Is No Simple Answer to This Question
- Collective Intelligence Has Defined the Role of mRNA
- A Synopsis of Prof. Matthew Cobb’s Review of the Collective Effort
The “molecular world” of nucleic acids is divided into two domains, DNA and RNA. While DNA structure stores encoded genetic function, RNA is structurally and functionally far more diverse. Among the many types of RNA, mRNA is critically important as a transient intermediate between DNA “blueprint” and protein “end product.”
This critical role of mRNA, which is central to all life forms, begs the question: who discovered mRNA? The short answer is that its discovery is the result of contributions made by a community of scientists over many years. However, who those scientists were and what they contributed is part of a longer, more complex story. This blog will discuss key elements of that story, and will draw from an excellent “deep dive” into the scientific history of mRNA published by Matthew Cobb, pictured here, professor at the University of Manchester, UK. Readers interested in more details may consult the original article and primary sources cited therein.
1961 marks a watershed in the ever evolving definition of mRNA function. Since 1961, more than 66,000 articles have been indexed in PubMed as messenger RNA or mRNA. As evidenced by the chart shown here, this remarkable volume of scientific literature is continuing to increase with each passing decade.
In 1953, Watson & Crick suggested that the sequence of bases in a DNA molecule contain ‘genetical information.’ The question then became how that information turned into biological function, i.e. what the nature of the genetic code is and how it works. George Gamow, a cosmologist who in the summer of 1953 wrote to Watson & Crick suggesting a model that involved proteins being synthesized on the DNA molecule itself (see Gamow, 1954), was initially responsible for focusing attention to this problem. “This theoretical model was dismissed by Crick as a non-starter,” says Cobb, because Crick was convinced that protein synthesis instead took place in the cytoplasm and required RNA, based on findings reported in 1947 by Torbjörn Caspersson in Sweden.
Also in 1947, the first hypothesis on how RNA fit into gene function was published in a French-language article by André Boivin and Roger Vendrely in Paris, and was succinctly expressed in the editor’s English summary: “the macromolecular deoxyribonucleic acids govern the building of macro-molecular ribonucleic acids, and in turn, these control the production of cytoplasmic enzymes,” i.e. proteins. But how?
Until the mid-1950s, according to Cobb, the thought of what was occurring in the cytoplasm during protein synthesis was “blurred by lack of knowledge.” Although RNA-rich structures, known as microsomal particles, were identified in the cytoplasm in the 1950s, it was only in 1959 that they were first referred to as ‘ribosomes’ in published literature. Ribosomal RNA, exemplified here, was the only form of RNA to be clearly identified, and back then it was assumed that it might be the RNA intermediary between DNA and proteins that so many scientists were searching for.
In 1957, as part of a Society of Experimental Biology symposium titled The Biological Replication of Macromolecules, Francis Crick gave a talk at University College, London. Published the next year as On Protein Synthesis, this lecture became famous for its description of what Crick called the central dogma, which outlined a hypothesis for the transfer of genetic information inside the cell. As depicted here, he argued that although information transfer occurred from DNA → RNA → protein, it was not possible for information to be transferred from protein to DNA.
While it might seem as though Crick was hypothesizing the existence of mRNA, this was not the case. Cobb suggests that, like everyone else, he was still hobbled by lack of understanding about the nature and function of the ribosome. Crick assumed that each ribosome consisted of a common protein structure and a unique sequence of RNA, which acted as a ‘template’ for the synthesis of a particular protein. This view was based partly on the discovery by Paul Zamecnik’s group at Massachusetts General Hospital that, during protein synthesis, radiolabeled amino acids were initially found only in the ribosomes, strongly suggesting that amino acids had to pass through the ribosome to be combined into a protein. It therefore seemed likely that the RNA in the ribosome was the template upon which the protein was made.
In order to explain how each amino acid got to the ribosome, Crick hypothesized the existence of what he called ‘the adaptor,’ a small and highly unstable set of RNA molecules that would bring each amino acid to the ribosome, allowing the ribosome to make the protein. In 1958, Zamecnik et al. identified this RNA species, which eventually became known as transfer RNA (tRNA), exemplified here.
As Crick explained, there had to be at least two kinds of RNA in the cytoplasm: ‘template RNA’ located inside the ribosome, and ‘metabolic’ or ‘soluble RNA,’ which he suspected was synthesized by each type of ribosome and corresponded to the code on the template RNA. Cobb points out that “[n]either of these kinds of RNA corresponded in form, function, or location to what we now call mRNA, and even the brilliant mind of Francis Crick did not recognize the need for a third form of RNA.”
Early Sightings of mRNA
Cobb adds that, in retrospect, a number of results from the 1950s indicated the presence of a short-lived RNA intermediary produced by genes, which we now identify as mRNA. However, in each of these early possible sightings, the speculative conclusions were not clearly supported by the results, or the results were interpreted erroneously.
The first early sighting, says Cobb, was reported in 1950 at the University of Brussels by Jeener & Szafarz, who attempted to identify differential turnover in various RNA fractions, but were hampered by relatively unsophisticated techniques by today’s standards. Nevertheless, they prophetically hypothesized that RNA was synthesized in the nucleus and then passed into the cytoplasm, where it was integrated with “cytoplasmic particles” before disappearing.
In 1958, Jeener demonstrated that RNase prevented synthesis of bacteriophage (aka phage) proteins following infection of a bacterial cell by phage, concluding that “RNA with a rapid turnover… is a specific product of the infection, and plays a role in the synthesis of phage protein.” However, as outlined in the next section, it was not until 1960 that the “eureka” moment for mRNA would finally occur.
According to Cobb, the realization that genes produce a messenger molecule first occurred in Paris, during a 1957 sabbatical visit by Arthur Pardee to the Institut Pasteur in Paris. Pardee was working with Jacques Monod on the genetic basis of induction, in which bacteria begin to synthesize β-galactosidase (β-gal, shown here) when grown on a medium containing lactose. Mutant lac– bacteria could not grow on lactose unless they acquired the z+ gene, which coded for the β-gal enzyme. Pardee showed that when the z+ gene was transferred into a lac– bacterium, β-gal synthesis began within minutes. This implied that there was an immediate chemical “signal,” i.e. an unidentified messenger molecule, which they called “X,” that passed directly from the introduced gene to the host cell’s protein synthesis system.
Subsequently, says Cobb, Pardee, François Jacob, and Monod began to consider that induction was not a positive effect, but rather what they called a ‘de-repression’— in other words, β-gal synthesis was normally repressed, but the presence of lactose somehow released that repression. By the time they published the full version of their experiments and interpretation in 1959, they were calling the substance that acted on the repressor gene a ‘cytoplasmic messenger.’ However, they could not define how exactly the process worked, and most importantly what the messenger was made of.
In his biography, Sydney Brenner, who worked with Crick at the University of Cambridge, indicates that relations between the Insititut Pasteur and the Cambridge group were cordial, although the two teams were working on solving different problems. He recalls that “the Paris people were interested in regulation. We essentially were interested in the code. So we had a slightly different approach.” Those two approaches finally intersected on April 15, 1960, when a small group of researchers, including Crick and Jacob, gathered in Brenner’s rooms in King’s College, Cambridge, pictured here.
As the group chatted, Jacob explained that Pardee had recently done an experiment showing that the β-gal gene did not produce a stable ribosome, but only the transitory messenger molecule X. “At this point,” recalls Crick in his book, “Brenner let out a loud yelp—he had seen the answer.” Jacob vividly described this eureka moment in his autobiography:
“Francis and Sydney leaped to their feet. Began to gesticulate. To argue at top speed in great agitation. A red-faced Francis. A Sydney with bristling eyebrows. The two talked at once, all but shouting. Each trying to anticipate the other. To explain to the other what had suddenly come to mind. All this at a clip that left my English far behind.”
According to Cobb, Brenner and Crick had just realized that the mysterious messenger could explain results suggesting that following phage infection, bacteria produced a short-lived form of RNA with the same base composition as phage DNA, but different from host ribosomal RNA. The two Cambridge men immediately seized the possibility that this short-lived RNA was the mysterious Paris messenger, X. This would make the ribosome an inert structure in the cell—Crick described it as a reading head, like in a tape recorder.
I agree with Cobb’s statement that “[t]his tape recorder metaphor can look rather quaint to 21st century eyes, and may need explaining to today’s students, but at the time it was a cutting-edge analogy, using the latest technological developments to explain a new biological phenomenon”.
Imagining mRNA immediately prompted ideas on how to isolate it. Jacob and Brenner’s proposed experiment required the help of Matt Meselson and his ultracentrifuges at Caltech in Pasadena. The challenge was to determine whether the messenger involved the creation of new ribosomes, as Jacob and Monod had initially suspected, or instead consisted of a new transient form of RNA that simply employed the old host ribosomes to turn its message into protein. New vs. old molecules of interest were distinguished by incorporation of heavy (C13, N15, etc.) or corresponding light (C12, N14, etc.) isotopes that allowed differential centrifugation at 37,000 rpm in a Spinco Model L centrifuge, a picture of which can be seen at this Science History Institute webpage.
Brenner recalls that after a tense month in California, endlessly fiddling with the experimental conditions (the magnesium concentrations proved decisive), they finally got the experiment to work. As they had hoped, no new ribosomes appeared. Instead, a relatively small and transient RNA that had been copied from the phage DNA was associated with old ribosomes that were already present in the bacterial host. This was messenger RNA! The subsequent May 13, 1961 publication in Nature by Brenner, Jacob, and Meselson titled An Unstable Intermediate Carrying Information from Genes to Ribosomes for Protein Synthesis can be accessed here.
Other researchers were independently taking a different route to the same conclusion, Cobb says. Work by Robert Risebrough at Harvard convinced Jim Watson that protein synthesis took place through the action of transitory ‘template’ RNA molecules that were combined with ‘genetically non-specific’ ribosomes. Together with François Gros and Howard Hiatt of the Institut Pasteur, and Charles Kurland and Walter Gilbert from Harvard, Watson and Risebrough began a long series of experiments that revealed the presence of transitory RNA molecules in cells that were briefly exposed to a radiolabeled RNA precursor. Cobb says that in February 1962, Watson sent a telegram, depicted here, asking Brenner to withhold publication until the Watson group was ready. The resultant publication, also on May 13, 1961 in Nature, titled Unstable Ribonucleic Acid Revealed by Pulse Labelling can be accessed at this link.
In the meantime, according to Cobb, Jacob and Monod built on the unpublished results of the Brenner–Jacob–Meselson experiment to codify the potential roles of what they termed ‘messenger RNA’ in a long review article, which appeared in Journal of Molecular Biology in May 1961, the same month as the two Nature papers. This review, titled Genetic Regulatory Mechanisms in the Synthesis of Proteins, is accessible here.
Proof of mRNA Function
Following strong but indirect evidence, the function of mRNA and its existence needed to be directly proven through rigorous science. This key functional data would be provided by Marshall Nirenberg at the National Institute of Arthritis and Metabolic Diseases in Bethesda, Maryland. According to Cobb, Nirenberg kept a remarkable series of laboratory diaries, in which he noted his ideas and aspirations. At the end of November 1960, Nirenberg’s diaries were full of discussions about then known cell-free systems, the importance of messenger RNA, and the use of synthetic RNA as a key.
In August 1961, Nirenberg and his post-doc Heinrich Matthaei published an article in the Proceedings of the National Academy of Sciences, titled Dependence of Cell-Free Protein Synthesis in E. Coli Upon Naturally Occurring or Synthetic Polyribonucleotides. Briefly, their findings indicated that “[p]olyuridylic acid contains the information for the synthesis of a protein having many of the characteristics of poly-L-phenylalanine. This synthesis was very similar to the cell-free protein synthesis obtained when naturally-occurring template RNA was added…” They concluded that “[one] or more uridylic acid residues therefore appear to be the code for phenylalanine. Whether the code is of the singlet, triplet, etc. type has not yet been determined. Polyuridylic acid seemingly functions as a synthetic template or messenger RNA.”
In their concluding statement, these researchers suggested that by using synthetic or natural messenger RNA and this stable, cell-free E. coli system, it “may well [be possible to] synthesize any protein corresponding to meaningful information contained in added RNA.” This prophetic vision has proven true, as supported by the fact that there are currently more than 2,000 articles in PubMed indexed to [(E. coli) AND cell-free] AND protein synthesis.
Nirenberg shared the 1968 Nobel Prize in Physiology or Medicine with Robert Holley and Gobind Khorana “for their interpretation of the genetic code and its function in protein synthesis.” Much later and then at NIH, I had the opportunity to supply Nirenberg with synthetic oligonucleotides and remember him as a soft-spoken scholar and gentleman, who expressed amazement over the fact that these oligos could be automatically synthesized so quickly.
“Textbook authors, students, and Wikipedia editors generally like simple stories,” says Cobb, who adds that “a simple view of the history of mRNA would claim that Jacob and Monod named it, while Brenner, Jacob, and Meselson subsequently isolated it.” In reality, fundamental advances in science generally occur after different groups of investigators study scientific questions using various strategies and tools, collectively leading to insights that clarify what was previously problematic or puzzling. This kind of complexity allowed for the discovery of mRNA, as laid out in detail by Cobb and briefly sketched out above.
Cobb elegantly concludes that “we have the advantage of looking backwards, knowing the answer; the participants were peering into a foggy future, trying to reconcile contradictory evidence and imagine new experiments that could resolve the problem. Their collective insights and imaginations laid the basis for today’s understanding and tomorrow’s discoveries.”
Proceedings of a three-day mRNA-centric symposium in 2014 at Cold Spring Harbor Laboratory, including historical accounts of the discovery of mRNA, are available at this link. By clicking the videos button, readers have access to a large number of excellent presentations, including talks given by some of the aforementioned scientists who were directly involved, namely (in alphabetical order), Brenner, Gilbert, Meselson, Pardee, and Watson.
My personal favorite is the video talk by Sydney Brenner, partly because of its style but also because I had the opportunity to work with Sydney at Lynx Therapeutics, where “Sydney Sequencing”—properly referred to as Massively Parallel Signature Sequencing (MPSS)—was initially investigated in the late-1990s as an early type of mRNA transcriptome profiling.
Brenner shared the 2002 Nobel Prize in Physiology or Medicine with Robert Horvitz and John Sulston “for their discoveries concerning genetic regulation of organ development and programmed cell death.” Sydney Brenner, who died at age 92 on April 5, 2019, is rightly remembered in an obituary in the New York Times as “a decipherer of the genetic code.”