RNA Catalysis

Genes in eukaryotes are not always colinear with their mRNA and protein products, but instead are often interrupted by stretches of noncoding DNA called "intervening sequences" (abbreviated as "IVS’s") or "introns." The discovery of these remarkable split genes had been announced by Phil Sharp and his research group at M.I.T. (see Great Experiment: The Discovery of RNA Splicing) and by Rich Roberts and colleagues at Cold Spring Harbor Laboratory only five years before our 1982 publication. In those five years, a number of laboratories had found that split genes were initially transcribed in toto to give a colinear precursor RNA, which was subsequently processed to remove the intron and generate the functional RNA, be it messenger RNA, transfer RNA or ribosomal RNA (for review see GENES 2000: 23 Nuclear splicing and RNA processing). The question of the mechanism of this RNA splicing reaction then came to the forefront.

The State of the Field in 1982
Mechanistic analysis of RNA splicing, like any other biochemical reaction, required being able to reproduce the event outside the cellular environment. In vitro splicing of yeast tRNA precursors was first achieved in John Abelson’s lab (Peebles et al., 1979), paving the way for the description of the reaction as involving one enzyme (an endonuclease) that excised the intron, and a second enzyme (an ATP-dependent RNA ligase) that stitched together the two tRNA halves. My own laboratory and Ole Westergaard’s had observed transcription and splicing of Tetrahymena pre-rRNA in isolated nuclei and nucleoli (Carin et al., 1980; Zaug et al., 1980). By 1981 we had separated transcription from splicing (Cech et al., 1981), setting the stage for a biochemical dissection of the reaction mechanism and the identification of the responsible activity or activities.

Up till this point, all enzymes known were proteins , and the biochemistry textbooks of the period considered no other possibility. I therefore believed that the activities responsible for rRNA splicing would be protein enzymes, as in fact turned out to be the case for tRNA splicing. I even obtained an NIH grant to fund the purification of these hypothetical proteins under the title "Enzymatic Splicing of a rRNA Precursor." Yet our initial experiments, summarized in Figure 1, produced a very unexpected result: the pre-rRNA underwent IVS excision even when we used SDS-phenol extraction and protease incubations to denature and then destroy any proteins present in the sample. The reaction only required the addition of salts and nucleotides and did not require the addition of any cellular protein component. We concluded that the splicing activity "may be due to a splicing enzyme tightly bound to the pre-rRNA, or it may be a novel case of an RNA-mediated reaction that requires no protein" (Cech et al., 1981). This set the stage for the research described in the paper highlighted here.

The experiment
We spent considerable effort testing our hypothesis that a splicing protein remained tenaciously attached to the isolated pre-rRNA despite treatments that would destroy most proteins. Although all efforts failed to show the presence of such a protein, negative evidence provides shaky grounds for any firm conclusion. We therefore decided to test the alternative hypothesis: Tetrahymena pre-rRNA splicing might be a novel RNA-mediated reaction. Had we been aware of speculations by Francis Crick, Leslie Orgel, Alex Rich and Carl Woese in the 1960s regarding the plausible role of catalytic RNA in prebiotic evolution, we might have thought that our idea was not such a major paradigm shift. As it was, we were driven to the idea of RNA catalysis not because we were enamored by the concept, but because our data seemed to have eliminated the more conventional possible explanations.

To test for RNA self-splicing, we needed an artificial pre-rRNA transcript that had never been exposed to Tetrahymena proteins and was therefore not subject to the criticism that it might be associated with a proteinaceous splicing enzyme. In principle, chemical synthesis could solve such a problem, but chemical synthesis of an RNA of this size (>1000 bases) was not possible then and has not even been accomplished now, almost 20 years later. The next best approach, as shown in Figure 2, was to synthesize the RNA by in vitro transcription of a purified, recombinant DNA template using a well-defined enzyme, RNA polymerase, which could then be removed from the solution. If such an RNA underwent auto-splicing, it would be time to announce the unprecedented news that RNA could form an active site for its own splicing reaction.

Figure 2 shows the DNA template for in vitro transcription that was constructed by Kelly Kruger, a part-time research assistant, Julie Sands, an undergraduate, and Dan Gottschling, then a graduate student in the lab. 1981 was still relatively early for recombinant DNA technology, and our lab had limited expertise. Cloning an intron-containing rRNA gene segment behind a promoter for a bacterial RNA polymerase required about one year. (Similar projects are now carried out by a single undergraduate in the lab in two weeks!) Ironically, at the time the cloning was initiated, our purpose was to produce more pre-rRNA as a substrate for the hypothetical (protein) splicing enzyme(s). However, by the time the clone was ready, so many indications had accumulated that the splicing was not protein-based that we were hopeful that the RNA transcript of the cloned DNA might splice itself.

We grew E. coli containing the recombinant DNA plasmid, recovered the plasmid, and cleaved it with a restriction enzyme to produce an end-point for transcription. We then added purified E. coli RNA polymerase and ribonucleotides (ATP, GTP, UTP and CTP) to make an artificial, shortened version of the Tetrahymena pre-rRNA, which was then deproteinized to remove the polymerase. The purified RNA was then incubated with the chemical ingredients that we had previously identified as necessary for splicing (i.e., a magnesium salt and guanosine, one of the building blocks of RNA) and the RNA self-spliced! (see Figure 2, Kruger et al., 1982) The nucleotide positions along the RNA chain that underwent cleavage and ligation in this extremely simple in vitro system were exactly the same sites where splicing occurred in vivo (Kan and Gall, 1982), giving us confidence that the in vitro reaction had biological relevance. The excised intron subsequently converted itself into a covalently closed circular RNA, a reaction previously discovered by Paula Grabowski in her Ph.D. thesis work.

Speculations and their subsequent outcome
Our findings immediately suggested some features of the RNA catalysis that were confirmed by later experiments, often through findings of other research groups. The major speculations presented in the Discussion of Kruger et. al. (1982) are discussed below.

Although we had demonstrated self-splicing only in vitro , we argued that self-splicing rather than enzyme catalysis would also be the case in vivo. The best evidence for this came from evolutionary and structurally related introns of the same class, called group I introns, from other biological sources. Splicing of yeast mitochondrial group I introns in vivo was inhibited by mutations in the intron RNA (Netter et al., 1982; Weiss-Brummer et al., 1983) consistent with the intron playing some sort of an active role in its own splicing. Furthermore, splicing of a bacteriophage T4 group I intron in E. coli was affected similarly by mutations in vitro and in vivo (Schroeder et al., 1991).

We thought it likely that all of the cleavage-ligation activity resided in the IVS RNA portion, although we could not rule out contribution by the flanking exon RNA sequences. Subsequent work indeed confirmed that the IVS catalyzed both RNA cleavage and ligation and comprised the "ribozyme," the term we coined to describe an RNA that had catalytic activity. The most convincing evidence was Art Zaug’s later conversion of the IVS (without exons) into true multiple-turnover catalysts; these ribozymes performed RNA cleavage-ligation reactions on separate RNA substrates (Zaug et al., 1986; Zaug et al., 1986). In a subsection of the paper entitled "Enzymatic RNA?," we proposed that the RNA must have a precise structure with two or more domains of the RNA forming an active site for the RNA cleavage-joining reactions. Because the reaction had a specific requirement for the mononucleoside G (guanosine) and was not active with C, U or A, we also proposed that it created a specific binding site for the G cofactor. Such substrate-binding sites were universal for protein enzyme catalysis, and now we were proposing that an RNA enzyme would have similar properties. The precise structure of the catalytic intron RNA was established through international efforts, which included a successful model based on phylogenetic sequence comparisons, X-ray crystallography of one of the intron RNA’s domains, and finally the determination of the crystal structure of the entire active catalytic core at lower resolution as shown in Figure 3 (Cate et al., 1986; Michel and Westhof, 1990; Golden et al., 1998). The proposal of a specific binding site for G was confirmed when the site was located within the RNA structure in elegant work of Michel et al. (1989; Michel et al., 1989).

The very coining of the generic term "ribozyme" by Kruger et al. implied that we thought that catalytic RNAs would be found elsewhere. We speculated that mRNA precursors were unlikely to be self-splicing per se , but that they might undergo such a reaction when complexed with small nuclear (sn) ribonucleoprotein particles. Such a model would help explain why mRNA precursors required RNAs in the form of U1, U2, U4, U5 and U6 snRNAs in order to splice. In fact, U2 snRNA was later shown to bind and position the chemical attacking group for pre-mRNA splicing (the 2-OH of an intronic A; Parker et al., 1987), and the U2, U5 and U6 snRNPs appear to form the catalytic core for splicing (Madhani and Guthrie, 1994; Wassarman and Steitz, 1992).

What are much more striking are the candidates for catalytic RNAs that we failed to mention in our paper. There was no mention of RNase P, an enzyme that cleaves a leader sequence from transfer RNA precursors and is unusual in that the enzyme is composed of both an RNA and a protein subunit. Within a year, Sydney Altman, in a research collaboration with Norman Pace, found that the RNA subunit of RNase P could catalyze pre-tRNA processing at rates similar to those of the holoenzyme (Guerrier-Takada et al., 1983). The RNase P discovery was key because it extended the concept of RNA catalysis to a true multiple-turnover enzyme. The other glaring omission was the ribosome, the RNA-protein complex responsible for reading messenger RNA and synthesizing proteins in all living organisms. Here, I must confess that I was unaware of Harry Nollers work implicating the RNA component of the ribosome as being particularly important for the peptidyl transfer reaction (Noller et al., 1972), an idea for which there is now direct evidence stemming from X-ray crystallographic structure determination (Nissen et al., 2000).

General significance of the paper as seen today
What was the impact of our paper? First, it spurred the search for other ribozymes. Within a few years, the list had grown to include RNase P, many other group I introns, the structurally distinct group II introns, and a number of small self-cleaving RNA motifs including the hammerhead ribozyme. This list of natural catalytic RNAs was ultimately extended by a large and diverse set of non-natural ribozymes identified through in vitro selection and amplification methods. Second, the availability of RNAs whose three-dimensional structures were critical for the reactions they catalyzed spurred the RNA structure field, both by providing excellent RNA systems and by increasing the incentive for structural information.

Finally, having an authentic example of RNA catalysis rekindled much earlier speculations about the role of RNA in the origins of life, speculations that had been withering for lack of any real examples. If the two most basic characteristics of a living system are information storage and ability to replicate that information, then RNA emerged as the one macromolecule that could achieve both functions — information and action, genotype and phenotype. Thus, perhaps in prebiotic times, life began with RNA replicating itself, with proteins and DNA making their entrances at later times (Gesteland, cech, and Atkins, 1999).

The Author
Born in Chicago, Illinois, Tom Cech was raised and educated in Iowa (B.A. in Chemistry from Grinnell College). He obtained his Ph.D. in Chemistry from the University of California, Berkeley and then engaged in postdoctoral research in the Department of Biology at M.I.T. in Cambridge, Massachusetts. In 1978 he joined the faculty of the University of Colorado, Boulder, where he became a Howard Hughes Medical Institute Investigator in 1988 and Distinguished Professor of Chemistry and Biochemistry in 1990.

Dr. Cech’s work has been recognized by many national and international awards and prizes, including the Heineken Prize of the Royal Netherlands Academy of Science (1988), the Albert Lasker Basic Medical Research Award (1988), the Nobel Prize in Chemistry (1989), and the National Medal of Science (1995). In 1987 Dr. Cech was elected to the National Academy of Sciences and also awarded a lifetime Professorship by the American Cancer Society.

Since 2000 Dr. Cech has been president of the Howard Hughes Medical Institute, headquartered in Chevy Chase, Maryland. He continues research on ribozyme structure and on telomerase in his Boulder, Colorado laboratory.