It is thought that the earliest forms of life on Earth were RNA molecules. According to the “RNA World” hypothesis, catalysts made entirely of RNA acquired the ability to replicate themselves in a way that involved Watson-Crick basepairing, thus ensuring the passage of genetic information to the progeny and genetic continuity of such self-replicative ribozymes. Today, relics of primordial RNA catalysts still play crucial roles in modern living organisms. A large body of evidence suggest that snRNAs, the RNA components of the spliceosome (the cellular machinery that performs the splicing reaction), belong to this category.

It has been estimated that at any given time there are around 150,000 mRNAs in each mammalian cell. Since the average human pre-messenger RNA has about 7-8 introns, and considering the rapid rate of turnover of the cellular mRNA pool, it is hardly surprising that a major portion of cellular resources are devoted to ensure the accuracy and speed of splicing reactions. Indeed, each cell contains roughly a million copies of each of the snRNPs (small nuclear ribo-nucleoproteins), the basic functional subunit of the spliceosome, which perform the removal of introns from pre-messenger RNAs.

Spliceosomes are highly complicated machines: they contain close to 200 different components that assemble in a highly dynamic and elaborate fashion to perform the splicing reaction. Despite their critical role in cellular physiology and their involvement in many human diseases, the mechanism of function of this complex cellular assembly remains largely unknown. This is mostly due to the daunting complexity of the spliceosome, which prevents the traditional biology methods from being applied to its study.

To solve this problem, and at the same time, to gain insight into the mechanism of human pre-mRNA splicing, we took a highly minimalistic approach. Previous studies had shown a critical role for the spliceosomal snRNAs in splicing catalysis, and similarities to ribozymes found in lower eukaryotes suggested that the spliceosome, also, might be an RNA enzyme. We chose just two of the snRNAs (out of over 200 RNA and protein factors in the spliceosome) which were shown to be at the heart of the active spliceosomes and tried to reconstitute the catalytic active site of the spliceosome in vitro. We could show that these two RNAs, U6 and U2, are inherently able to form the basepaired structure they form in vivo without the help of any additional factors. Thus, the ability to form the functionally active structure is embedded in the sequence of these two RNAs.

Next, we tested them to see if they show biologically relevant activity, that is, if they can perform splicing. A number of activity assays indicated that the U6/U2 basepaired complex could indeed show catalytic activity that was related to the splicing reaction, but differed from the authentic splicing reaction. Very recently, we succeeded in obtaining splicing-like catalysis from these two RNAs: upon incubation with suitable pre-mRNA-like substrates, the U6/U2 basepaired complex catalyzed two consecutive reactions on the substrates that resembled the first and the second steps of splicing.