Pre-mRNA splicing is a key step in mRNA maturation. Organisms acquired mechanisms to faithfully join exons to produce functional proteins and to allow a certain plasticity to make alternative use of exons to produce multiple mRNA isoforms from a single gene.
In metazoan, most genes are comprised of multiple exons that are interspersed by relatively large introns of a few hundred up to tens of kilo bases. In such a gene architecture, it is believed that each exon boundary is recognised and paired by splicing machinery prior to the splicing of introns. If splicing happens at once on an mRNA, the pairing of exon boundaries needs to take place once. On the other hand, if splicing happens sequentially, a new set of exon boundaries need to be recognised for the subsequent reaction to happen. At present, we do not know to what extent splicing occurs sequentially and what could be the mechanism to ensure the correct pairing of exon boundaries during the multi-step splicing reaction.
In order to monitor the order of splicing in individual genes, we aim to capture nascent transcripts from the nuclear fraction and sequence their full-length using a recently-emerged long-read sequencing platform called Nanopore. The project involves tissue culture, biochemical isolation of nuclei and nascent transcripts, operation of Nanopore sequencer and the accompanying computational analysis.