When RNA goes off-script: misadventures in transgene expression
Despite the ubiquitous nature of RNA processing, researchers using plasmid models often assume the transcripts expressed in cells will precisely match their designed DNA sequences. This assumption is rarely tested and can be invalidated by routine sequence changes to the plasmid DNA. Spurious RNA processing can generate compelling-but-incorrect results that misrepresent the system's true biology. These errors can mislead researchers (and even entire fields) until substantial time and effort are invested to uncover the truth. To test whether various synthetic sequences participate in aberrant splicing, we designed a splicing reporter based on mCherry split by a poorly spliced (about 50 percent in HEK293T cells) intron derived from COQ8B intron 6. We inserted a small library of test sequences into this reporter plasmid (e.g. HA and V5 epitope tags), pooled and transfected the plasmids, and then performed RNA-seq to determine splicing outcomes.