"In Trans" Exon Ligation: A New System for Studying the Second Step of Pre-mRNA Splicing

A long-term goal of my research is to elucidate the catalytic mechanisms and active site structures of group II self-splicing introns and the mammalian spliceosome. Introns are incoherent nucleotide sequences that interrupt the coding regions of genes. They are removed from newly made RNA transcripts by a process called RNA splicing. Since most genes in multicellular organisms contain introns, their timely and precise removal is an essential biochemical process. Amazingly, some introns are capable of removing themselves. These so called "self-splicing" introns, of which group II introns are one class, fold into conserved tertiary structures which confer catalytic activity to the RNA itself. Most introns in eukaryotic nuclei, however, require the action of a spliceosome for their excision. The spliceosome is a complex macromolecular assemblage containing five small stable RNAs and a multitude of proteins, not all of which have been identified. Interestingly, the chemical mechanisms (the reactions and their temporal order; see figure) for intron excision by both group II introns and the spliceosome are identical. Because of this, it is widely believed that the two are evolutionarily related. However, because detailed functional and structural pictures are largely lacking for either system, this hypothesis is highly debatable at present. In an effort to shed light on this debate, ongoing work in my laboratory is aimed at elucidating the mechanistic and structural similarities and differences between these two systems.

As a starting point, we have chosen to concentrate on the exon ligation step. In particular, we are working to develop new assays for both the spliceosome and group II systems in which the 3' splice site/exon boundary can be added in trans to the catalytic core. Such physical separation of enzyme and substrate will make it possible to determine detailed kinetic rate profiles for exon ligation in each system, which in turn allow one to distinguish between substrate functional groups required for binding versus those required for catalysis. Incorporation of site-specific cross-linking reagents into the substrate will additionally facilitate active site structural elucidation.

In the past year, we have completed development of such an "in trans" exon ligation system for the spliceosome. Using RNA substrates that contain sequences required for the first step of splicing, but not those required for the second, we can now accumulate spliceosomes that have catalyzed 5' splice site cleavage and lariat formation, but cannot advance further because they lack a viable 3' splice site. If a second RNA that contains a 3' splice site is subsequently supplied, however, a single round of exon ligation is initiated.

This new system has opened a unique window into the exon ligation reaction, and we have already encountered several surprises. Perhaps the most striking regards the function of the polypyrimidine tract (PPT) in definition of the 3' splice site. Most mammalian introns contain a 10-40 nucleotide tract of pyrimidines (U's and C's) between the branch site and 3' splice site. It is well-established that this tract is important for branch site definition prior to lariat formation. Yet many studies have also suggested that the polypyrimidine tract additionally functions to help define the 3' splice site prior to exon ligation. Because of its first step requirement, however, it was previously impossible to dissect out the exact PPT requirement for the second step. In our system, the branch site and 3' splice site are on separate RNA molecules. By successively truncating the 3' splice site substrate, we have now shown that there is actually little or no polypyrimidine tract requirement for 3' splice site definition. Our data are consistent with a scanning mechanism for 3' splice definition.