Single Molecule Imaging In Vivo Determines Post-Transcriptional RNA Processing Dynamics

The synthesis and splicing of pre-mRNA to form completed transcripts requires coordination between two large multi-subunit complexes (the RNA polymerase and the spliceosome). Using dual color single molecule RNA imaging in living human cells, we previously observed kinetic competition during the transcription cycle which resulted in both co- and post- transcriptional splicing of pre-mRNA. By combining transcription site fluctuation analysis and RNA imaging we construct a model of RNA synthesis and processing. We see that the majority of pre-mRNAs are spliced after release, while diffusing away from the site of transcription. By single molecule tracking, we measure the distribution of pre-mRNA around the site of transcription. By Raster Image Correlation Spectroscopy (RICS), we measure the diffusion coefficient of pre-mRNA and determine that pre-mRNA is spliced within 5 seconds of cleavage from the transcription site suggesting that intron removal is more efficient after cleavage and release of nascent RNA. We further investigate the role of splicing factors and mutations on the transcription and splicing dynamics and localization of pre-mRNA.Left: intron, exon and merge (black arrow indicates the transcription site). Right: profile through an unspliced and spliced RNA molecule.View Large Image | View Hi-Res Image | Download PowerPoint Slide