1. Non-invasive and high-throughput interrogation of exon-specific isoform expression
- Author
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Milica Živanić, Sigrid C. Schwarz, Tabea Strauß, Peter Heutink, Bianca Eßwein, Dominic Schwarz, Martin Zirngibl, Marcus Conrad, Christian Grätz, Francesco Leandro Vaccaro, Luisa Krumwiede, Julian Geilenkeuser, Wolfgang Wurst, Simone Göppert, Sebastian Doll, Florian Giesert, Christoph Gruber, Günter U. Höglinger, Tobias Santl, Gerald Raffl, Eva Magdalena Beck, Gil G. Westmeyer, Maren Beyer, Valentin Evsyukov, Dong-Jiunn Jeffery Truong, Enikő Baligács, Deniz Tümen, Johann Dietmar Körner, Niklas Armbrust, Teeradon Phlairaharn, and Eva-Maria Lederer
- Subjects
Gene isoform ,Proteomics ,CRISPR-Cas systems ,RNA splicing ,Proteome ,RNA Stability ,Induced Pluripotent Stem Cells ,MAPT protein, human ,tau Proteins ,Biology ,metabolism [RNA-Binding Proteins] ,metabolism [RNA, Messenger] ,Exon ,genetics [RNA, Messenger] ,Technical Report ,Protein splicing ,ddc:570 ,Humans ,Protein Isoforms ,FOXP1 protein, human ,genetics [RNA-Binding Proteins] ,RNA, Messenger ,Induced pluripotent stem cell ,Synthetic biology ,metabolism [Repressor Proteins] ,MBNL1 protein, human ,metabolism [Forkhead Transcription Factors] ,Alternative splicing ,Biological techniques ,High-throughput screening ,RNA-Binding Proteins ,Forkhead Transcription Factors ,Cell Biology ,FOXP1 ,Exons ,Embryonic stem cell ,metabolism [tau Proteins] ,Cell biology ,High-Throughput Screening Assays ,metabolism [Induced Pluripotent Stem Cells] ,Repressor Proteins ,Alternative Splicing ,genetics [Repressor Proteins] ,genetics [tau Proteins] ,HEK293 Cells ,genetics [Forkhead Transcription Factors] ,CRISPR-Cas Systems ,Single-Cell Analysis - 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., Truong et al. developed a cell-based reporter system, EXSISERS, that enables non-invasive quantification of the protein expression levels of exon-specific isoforms via intein-mediated protein splicing.
- Published
- 2021
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