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.
Despite their fundamental role in assessing (patho)physiological cell states, conventional gene reporters can follow gene expression but leave scars on the proteins or substantially alter the mature messenger RNA. Multi-time-point measurements of non-coding RNAs are currently impossible without modifying their nucleotide sequence, which can alter their native function, half-life and localization. Thus, we developed the intron-encoded scarless programmable extranuclear cistronic transcript (INSPECT) as a minimally invasive transcriptional reporter embedded within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in the mature endogenous RNA without sequence alterations and an additional engineered transcript that leaves the nucleus by hijacking the nuclear export machinery for subsequent translation into a reporter or effector protein. We showcase its use in monitoring interleukin-2 (IL2) after T cell activation and tracking the transcriptional dynamics of the long non-coding RNA (lncRNA) NEAT1 during CRISPR interference-mediated perturbation. INSPECT is a method for monitoring gene transcription without altering the mature lncRNA or messenger RNA of the target of interest.
Despite their pivotal role in evaluating (patho)physiological cell states, traditional gene reporters can follow gene expression but leave marks on proteins or significantly modify mature mRNA. Multi-time point readouts of non-coding RNAs are, to date, not applicable without changing their nucleotide sequence, which may affect their natural function, half-life, and localization. We thus created INSPECT (Intron-encoded Scarless Programmable Extranuclear Cistronic Transcripts) as a minimally invasive transcriptional reporter nested within an intron of a gene of interest. Post-transcriptional excision of INSPECT results in a mature RNA with no sequence changes and an additional synthetic transcript that leaves the nucleus by hijacking the nuclear host export machinery for cytosolic translation into a reporter protein. Here, we first illustrate the cloning of such an INSPECT DNA donor construct and an auxiliary CRISPR/Cas9 plasmid to insert INSPECT into a gene of interest and, secondly, exemplary for NEAT1_long, we showed the generation of such a cell line.
Exon-specific isoform expression from alternatively spliced mRNA is a fundamental mechanism that substantially enriches a cell’s proteome. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression over multiple time points and at the protein level. We thus developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms by scarlessly excising reporter proteins from the nascent polypeptide chain via highly efficient, intein-mediated protein splicing. EXSISERS enables non-invasive monitoring of exon-specific isoform expression with high sensitivity and cellular resolution and enables high-throughput screening of exon-specific therapeutic interventions.This protocol on how to generate EXSISERS reporter cell lines is related to the publication ‘Non-invasive and high-throughput interrogation of exon-specific isoform expression’ in Nature Cell Biology.
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