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Non-invasive and high-throughput interrogation of exon-specific isoform expression
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Non-invasive and high-throughput interrogation of exon-specific isoform expression
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Non-invasive and high-throughput interrogation of exon-specific isoform expression
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Journal Article
Non-invasive and high-throughput interrogation of exon-specific isoform expression
2021
Overview
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.
Publisher
Nature Publishing Group UK,Nature Publishing Group
Subject
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/ Analysis
/ Biomedical and Life Sciences
/ Exons
/ Forkhead Transcription Factors - genetics
/ Forkhead Transcription Factors - metabolism
/ High-Throughput Screening Assays
/ Humans
/ Induced Pluripotent Stem Cells - metabolism
/ Isoforms
/ Methods
/ mRNA
/ Proteins
/ Proteome
/ Repressor Proteins - genetics
/ Repressor Proteins - metabolism
/ RNA-Binding Proteins - genetics
/ RNA-Binding Proteins - metabolism
/ Splicing
