Cech found that the nuclear ribosomal RNA from Tetrahymena contains an intervening sequence. In efforts to build an in vitro system in which splicing would occur, he placed unspliced rRNA in reactions containing and lacking extracts prepared from cells. Amazingly, the control reactions for the splicing reactions, those lacking the added extract, also spliced out the intervening sequence. Naturally, contamination was suspected, and strenuous efforts were made to remove any possible Tetrahymena proteins from the substrate RNA, but splicing in the absence of Tetrahymena extract persisted. Finally, Cech placed the gene for the rRNA on a plasmid that could replicate in E. coli, prepared the DNA from E. coli cells, a situation that had to be devoid of any hypothetical Tetrahymena protein, and he still found splicing. This proved, to even the most skeptical, that the Tetrahymena rRNA was performing splicing on itself without the action of any Tetrahymena proteins.

As shown in Fig. 1, guanosine is essential to this self-splicing reaction, but it does not contribute chemical energy to the splicing products. Further studies on the Tetrahymena self-splicing reaction show that not only is a 480-nucleotide section of RNA removed from the middle of the ribosomal RNA, but the removed portion then goes on to close on itself forming a circle and releasing a short linear fragment. At first it seems surprising that neither ATP nor any other energy source is required for the cutting and splicing reactions. The reason is that external energy is not required. Chemically, all the reactions are transesterifications and the numbers of phosphodiester bonds are con served. One might ask, then, why the reaction proceeds at all. One answer is that in some cases the products of the reactions are three polynucleotides where initially only one plus guanosine existed before. Together these possess higher entropy than the starting molecules, and therefore their creation drives the reaction forward.

Fig1. The series of self-splicing reactions followed by Tetrahymena rRNA.

The transesterification reactions involved in the self-splicing proceed at rates many orders of magnitude faster than they could with ordinary transesterifications. Only two reasons can explain the stimulated rate of these reactions. First, the secondary structure of the molecules can hold the reactive groups immediately adjacent to one another. This increases their effective collision frequencies far above their normal solution values. The second reason is that the probability of a reaction occurring with a collision can be greatly improved if the bonds involved are strained. Studies with very small self–cutting RNAs and also molecular dynamics calculations indicate that such a strain is crucial to the reactions. Undoubtedly, self–splicing utilizes both principles.

Self–splicing has been found in two of the messenger RNAs of the bacteriophage T4, and in the splicing of mRNA in the mitochondrion of yeast. The mitochondrial self–splicing introns comprise a second group of self–splicing introns. Their secondary structure differs from those of the Group I self–splicing introns of which the Tetrahymena rRNA intron is an example. The Group II introns do not use a free guanosine to initiate the splicing process. They use an internal nucleotide, and in this respect use a reaction mechanism more like that used in processing pre–mRNA. The existence of splicing in bacteria and eukaryotes suggests that splicing in general has existed in the common precursor to both organisms. The scarcity splicing events in prokaryotes might be a result of the greater number of generations they have had in which to select for the loss of introns. Eukaryotes may still be struggling with the “infection.”