Investigating the role of the U2 and U6 snRNAs in exon ligation during pre-mRNA splicing

The DNA of a cell is copied into a pre-messenger RNA (pre-mRNA) that the cell uses as a template for protein production. All the information contained in DNA is not required for making proteins, therefore, unwanted information must be removed before a protein is made. The unwanted information is removed, or 'spliced', from pre-mRNA by a process similar to the editing of unwanted frames from a film. This 'splicing' of the pre-mRNA is very important because it must occur accurately in order for functional proteins to be produced. A mistake by even one nucleotide could have disastrous effects on the final protein produced. The process of 'splicing' is carried out by a large RNA/protein complex called the spliceosome. The spliceosome interacts with the pre-mRNA to identify and 'splice' out the unwanted regions. An increasing amount of evidence points to the RNA components of the spliceosome as being critical for the actual cutting process of splicing. The work that will be undertaken during the tenure of this research grant will address how the RNA components of the spliceosome interact with each other and the pre-mRNA to identify then 'splice' out the unwanted regions. This is basic research that will contribute to our knowledge of a critical cellular process.

Transcription in eukaryotic cells produces pre-messenger RNAs (pre-mRNAs) that contain intron regions that are removed by the process of pre-mRNA splicing. Accuracy of splicing is critical for production of functional mRNAs and subsequent synthesis of proteins that define and control cell behaviour. The splicing of intron sequences from pre-mRNA and ligation of coding exon sequences is carried out by the spliceosome, a large RNA/protein complex. It is almost certain that splicing is catalysed by the snRNA components of the spliceosome, therefore, it is important to determine how the snRNAs function in splicing. The correct alignment of the exon ends for ligation is a critical step in splicing. During splicing the exon ends are aligned precisely with U5 snRNA loop 1 for ligation. It is unclear how the many different exons are each aligned precisely with U5 loop 1 for ligation during the second step of splicing. This proposal will investigate, by cross-linking and analysis of U2 and U6 snRNA mutants, how the U2 and U6 snRNAs direct the exons to U5 loop 1 for ligation during splicing. The correct assembly of the snRNAs with the pre-mRNA is also important for splicing. This proposal will investigate by cross-linking analysis a novel interaction of the U2 snRNA with the 5' exon of the pre-mRNA. Investigating this novel interaction will provide important information on how the snRNAs assemble with the pre-mRNA to carry out splicing. Overall, this proposal will provide insight into how the spliceosome can bring together different exon sequences to produce functional mRNA following intron removal and will further our knowledge of the RNA interactions required for pre-mRNA splicing.