Splicing of nuclear pre-mRNA involves formation of a multicomponent complex termed the spliceosome in which the splicing reaction takes place. Small nuclear ribonucleoprotein particles (snRNPs) U1, U2, U4, U5 and U6 are involved in splicing and are found associated with specific splicing complexes. Since recognition of the 5´ splice site (5´SS) is required for spliceosome assembly it is important to elucidate the nature and temporal order of interactions between the 5´SS sequence and the spliceosome components. To this end, we have used an in vitro system in which a short RNA oligonucleotide comprising the 5´SS consensus sequence is recognized by at least two distinct factors: first by the 5´ end of U1 snRNA and subsequently by a component(s) of U2/U4/U5/U6 snRNP complex (the spliceosome). In vitro, spliceosome assembly involves an intermediate complex in which the 5´SS RNA oligo binds to U4/U5/U6 snRNP in the absence of U2 snRNP. The specificity of this association is remarkably similar to that observed in vivo as defined by the 5´SS consensus sequence. Thus, recognition of the 5´SS by the U4/U5/U6 snRNP complex defines the functional 5´ splice site. We have recently demonstrated that the most highly conserved element of the 5´SS consensus, the GU dinucleotide at the 5´ end of the intron is recognized by p220, a protein component of U5 snRNP present in the spliceosome. The high degree of specificity of p220 recognition of the 5´SS is remarkable. This recognition represents the first example of a highly specific protein:5´SS RNA interaction within the spliceosome. In the presence of a second RNA containing a 3´SS region, the short 5´SS RNA oligo undergoes both the first and the second steps of splicing. Despite of the simplicity of this system, this trans-splicing is similar to the standard cis-splicing reaction with regard to the specificity of substrate recognition. This assay offers a powerful method for detailed studies of the mechanism of splicing.

Our interest in studying interactions between snRNP particles stems from a prediction that snRNAs play a crucial role in the catalysis of splicing. Hopefully, information gained from the structural analysis of splicing complexes will help us to understand the mechanism of pre-mRNA splicing and the role of snRNA sequences in this process.

An independent line of research concerns the replication of RNA by DNA-dependent RNA polymerases. We have chosen replication of hepatitis delta virus (HDV) RNA to study this process. Using an in vitro system based on nuclear extracts of HeLa cells we have shown that RNA polymerase II directs site-specific initiation of transcription from HDV RNA templates. We are currently studying RNA requirements that specify the promoter activity of RNA templates as well as the protein components other than RNA polymerase II that are involved in this process. In addition, we are interested in the role of delta antigen, the only polypeptide encoded by the HDV genome. We have constructed several mutants of HDV that affect the delta antigen reading frame and have shown that this results in a temperature-sensitive phenotype for HDV replication. Structural analysis of delta RNA and studies on the mechanism of its replication may provide further insights into the general question of the origin and evolution of viroidlike RNA pathogens and help to identify other members of this group.