Disambiguating Transcriptional Effects from Post-Transcriptional Effects of Genetic Variants with an Inferred Stability Optimized Massively Parallel Reporter Assay (ISOMPRA).

Some widely used methods to study functional effects of DNA sequence on transcription cannot readily disambiguate these from post-transcriptional impacts of the same element on RNA. Therefore, we developed an approach enabling assessment of DNA/RNA regulatory elements in parallel: Inferred Stability Optimized Massively Parallel Reporter Assay (ISOMPRA). ISOMPRA quantifies pre-spliced RNA to more directly assess transcription rate, and spliced RNA, corrected for transcription rate, to correctly identify RNA stability-altering 3' UTR elements. We benchmark ISOMPRA with both predicted enhancers and 3' UTR elements, selected regions containing autism patient mutations. We identify numerous functional enhancers/UTRs, and discover a subset with high impact mutations. We further show failing to consider both potential transcriptional and post transcriptional effects results in numerous false-positives and false-negatives. Finally, we adapt ISOMPRA for in vivo analysis of element effects via AAV transduction. Combined with Cre-dependent priming sites, this permits analyses of both transcriptional and post-transcriptional effects within specific cell types of the brain, and further enables measurement of regulatory impacts of in vivo traits, such as sex. Overall design: RNA sequencing libraries prepared from mouse RNA and HEK293 cell RNA alongside plasmid and AAV sequenced DNA