Non-invasive and high-throughput interrogation of exon-specific isoform expression

  • Nat Cell Biol. 2021 Jun;23(6):652-663. doi: 10.1038/s41556-021-00678-x.
Dong-Jiunn Jeffery Truong  1  2 ,  Teeradon Phlairaharn  1  2 ,  Bianca Eßwein  3 ,  Christoph Gruber  3 ,  Deniz Tümen  1  4 ,  Enikő Baligács  1  2 ,  Niklas Armbrust  1  2 ,  Francesco Leandro Vaccaro  1  2 ,  Eva-Maria Lederer  1  2 ,  Eva Magdalena Beck  1  2 ,  Julian Geilenkeuser  1  2 ,  Simone Göppert  1  2 ,  Luisa Krumwiede  1  2 ,  Christian Grätz  1  2 ,  Gerald Raffl  1  2 ,  Dominic Schwarz  1  2 ,  Martin Zirngibl  1  2 ,  Milica Živanić  1  2 ,  Maren Beyer  1  2 ,  Johann Dietmar Körner  1  2 ,  Tobias Santl  1  2 ,  Valentin Evsyukov  5  6  7 ,  Tabea Strauß  5  6 ,  Sigrid C Schwarz  5  6 ,  Günter U Höglinger  5  6  7 ,  Peter Heutink  8  9 ,  Sebastian Doll  10 ,  Marcus Conrad  10  11 ,  Florian Giesert  3 ,  Wolfgang Wurst  3  5  12 ,  Gil Gregor Westmeyer  13  14
Affiliations
  • 1. Institute for Synthetic Biomedicine, Helmholtz Zentrum München, Oberschleißheim, Germany.
  • 2. Department of Chemistry and TUM School of Medicine, Technical University of Munich, Munich, Germany.
  • 3. Institute of Developmental Genetics, Helmholtz Zentrum München, Oberschleißheim, Germany.
  • 4. Department of Internal Medicine I, University Hospital Regensburg, Regensburg, Germany.
  • 5. German Center for Neurodegenerative Diseases (DZNE), Munich, Germany.
  • 6. Department of Neurology, Technical University Munich, Munich, Germany.
  • 7. Department of Neurology, Hannover Medical School, Hannover, Germany.
  • 8. Department for Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany.
  • 9. German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
  • 10. Institute of Metabolism and Cell Death, Helmholtz Zentrum München, Oberschleißheim, Germany.
  • 11. Laboratory of Experimental Oncology, National Research Medical University, Moscow, Russia.
  • 12. TUM School of Life Sciences, Technical University of Munich, Freising, Germany.
  • 13. Institute for Synthetic Biomedicine, Helmholtz Zentrum München, Oberschleißheim, Germany. [email protected].
  • 14. Department of Chemistry and TUM School of Medicine, Technical University of Munich, Munich, Germany. [email protected].
PMID: 34083785 DOI: 10.1038/s41556-021-00678-x
Abstract

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

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