Ramon Y. Birnbaum, Rupali P. Patwardhan, Mee J. Kim, Gregory M. Findlay, Beth Martin et al.
In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types.
Ramon Y. Birnbaum, Rupali P. Patwardhan, Mee J. Kim, Gregory M. Findlay, Beth Martin, Jingjing Zhao, Robert J. A. Bell, Robin P. Smith, Angel A. Ku, Jay Shendure, Nadav Ahituv. Systematic Dissection of Coding Exons at Single Nucleotide Resolution Supports an Additional Role in Cell-Specific Transcriptional Regulation. 2014-10-23. https://doi.org/10.1371/journal.pgen.1004592
The SORL1 exon 17 eExon library contained 16,483 distinct mutant haplotypes and 16,582 tagged constructs and covered all 1,665 possible single-nucleotide substitutions. The tagged pGL4.23 library was delivered to mouse liver by hydrodynamic tail-vein injection and RNA-derived tags were sequenced 24 hours later.
The SORL1 exon 17 eExon library contained 16,483 distinct mutant haplotypes and 16,582 tagged constructs and covered all 1,665 possible single-nucleotide substitutions. The tagged pGL4.23 library was transiently transfected into HeLa cells and RNA-derived tags were sequenced 24 hours later.
The PPARG exon 6 eExon library contained 22,320 distinct mutant haplotypes and 22,506 tagged constructs and covered all 1,614 possible single-nucleotide substitutions. The tagged pGL4.23 library was transiently transfected into HeLa cells and RNA-derived tags were sequenced 24 hours later.
The TRAF3IP2 exon 2 eExon library contained 10,029 distinct mutant haplotypes and 10,071 tagged constructs and covered all 1,962 possible single-nucleotide substitutions. The tagged pGL4.23 library was transiently transfected into HeLa cells and RNA-derived tags were sequenced 24 hours later.
The PPARG exon 6 eExon library contained 22,320 distinct mutant haplotypes and 22,506 tagged constructs and covered all 1,614 possible single-nucleotide substitutions. The tagged pGL4.23 library was delivered to mouse liver by hydrodynamic tail-vein injection and RNA-derived tags were sequenced 24 hours later.
The TRAF3IP2 exon 2 eExon library contained 10,029 distinct mutant haplotypes and 10,071 tagged constructs and covered all 1,962 possible single-nucleotide substitutions. The tagged pGL4.23 library was delivered to mouse liver by hydrodynamic tail-vein injection and RNA-derived tags were sequenced 24 hours later.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 6 files (ZIP)README.txtSRA_SRP044727_runinfo.csvTable_S2_exons_tested_for_enhancer_activity.xlsxTable_S3_eExon_library_characteristics.xlsxTable_S4_mutation_effect_size_summary.pdfTable_S5_summary_all_SNVs_MPRA.xlsx