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

Alternative Splicing CRISPR-Cas Systems Exons Forkhead Transcription Factors / genetics HEK293 Cells High-Throughput Screening Assays Humans Induced Pluripotent Stem Cells / metabolism Protein Isoforms Proteome Proteomics RNA Stability RNA, Messenger / genetics RNA-Binding Proteins / genetics Repressor Proteins / genetics Single-Cell Analysis tau Proteins / genetics

Journal

Nature cell biology

ISSN: 1476-4679

Titre abrégé: Nat Cell Biol

Pays: England

ID NLM: 100890575

Informations de publication

Date de publication:
06 2021

Historique:

received: 06 01 2020

accepted: 01 04 2021

pubmed: 5 6 2021

medline: 24 8 2021

entrez: 4 6 2021

Statut: ppublish

Résumé

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.

Identifiants

DOI: 10.1038/s41556-021-00678-x PMID: 34083785 PMC: PMC8189919

pubmed: 34083785

doi: 10.1038/s41556-021-00678-x

pii: 10.1038/s41556-021-00678-x

pmc: PMC8189919

doi:

Substances chimiques

FOXP1 protein, human 0

Forkhead Transcription Factors 0

MAPT protein, human 0

MBNL1 protein, human 0

Protein Isoforms 0

Proteome 0

RNA, Messenger 0

RNA-Binding Proteins 0

Repressor Proteins 0

tau Proteins 0

Types de publication

Journal Article Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

Pagination

652-663

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