Transcriptional neighborhoods regulate transcript isoform lengths and expression levels

Sequence features of genes and their flanking regulatory regions are determinants of RNA transcript isoform expression and have been used as context-independent, plug-and-play modules in synthetic biology. However, genetic context, including the adjacent transcriptional environment, also influences transcript isoform expression levels and boundaries. We used synthetic yeast strains with stochastically repositioned genes to systematically disentangle sequence from contextual effects. Profiling 120 million full-length transcript molecules across 612 genomic perturbations, we observed sequence-independent alterations to gene expression levels and transcript isoform boundaries that were influenced by neighboring transcription. We identified features of transcriptional context that could predict these alterations and used these features to engineer a synthetic circuit where neighboring transcription controlled transcript length. This demonstrates how positional context can be leveraged in synthetic genome engineering.