One early difficulty in studying splicing of mRNA was obtaining the RNA itself. Cells contain large amounts of rRNA, but most of the mRNA has been processed by splicing. Further, only a small fraction of the unspliced pre–mRNA present at any moment is from any one gene. One convenient source of pre-mRNA for use in splicing reactions came from genetic engineering. The DNA for a segment of a gene containing an intervening sequence could be placed on a small circular plasmid DNA molecule that could be grown in the bacterium Escherichia coli and easily purified. These circles could be cut at a unique location and then they could be transcribed in vitro from special phage promoters placed just upstream of the eukaryotic DNA (Fig.1). By this route, large quantities of unspliced substrate RNA could be obtained.

Fig1. The use of SP6 or T7 phage promoters on small DNA molecules to generate sizeable amounts in vitro of RNAs suitable for study of splicing reactions.

Both of the two self-splicing reactions and the snRNP catalyzed splicing reactions can be drawn similarly (Fig.2). In the self-splicing cases a hydroxyl from a guanosine nucleotide or an adenine in the chain attacks the phosphodiester and a transesterification ensues in which the 5’ end of the RNA is released. For the Group I self-splicing reaction, a tail is formed, and for the Group II and mRNA splicing reactions, a ring with a tail is formed. Then a hydroxyl from the end of the 5’ end of the molecule attacks at the end of the intervening sequence and another transesterification reaction joins the head and tail exons and releases the intron.

Fig2. The two classes of self–splicing RNA and the pathway of nuclear mRNA splicing, all drawn to emphasize their similarities.

In the case of pre–mRNA splicing, the same reaction occurs, but it must be assisted by the snRNP particles. In some intervening sequences of yeast, internal regions are involved in excision and bear some resemblance to portions of the U1 sequences.

The similarities among the splicing reactions suggest that RNA was the original molecule of life since it can carry out the necessary functions on its own, and only later did DNA and protein evolve. The splicing reactions that now require snRNPs must once have proceeded on their own.