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

Dong‐Jiunn Jeffery Truong ,Günter U Höglinger,Tabea Strauß,Deniz Tümen,Milica Živanić,Bianca Eßwein,Enikő Baligács,Johann Dietmar Körner,Simone Göppert,Christian Grätz,Wolfgang Wurst,Christoph Gruber,Marcus Conrad,Peter Heutink,E Beck ,Sebastian Doll,Julian Geilenkeuser,Francesco Leandro Vaccaro,Niklas Armbrust,Moritz Beyer ,Valentin Evsyukov,Gerald Raffl,Teeradon Phlairaharn,Eva-Maria Lederer,Dominic Schwarz,Tobias Heinrich Santl ,Luisa Krumwiede,Sigrid C Schwarz,Florian Giesert,Martin Zirngibl,Gil G Westmeyer

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.

Highlights

Alternative splicing occurs in >90% of genes, and its impairment is associated with diseases such as spinal muscular atrophy and Parkinson’s disease
In the adult human central nervous system, tau is expressed in six isoforms produced by alternative splicing of microtubule-associated protein tau (MAPT) exons 2, 3 and 10 (Fig. 1a)
We found a linear relationship between the relative luminescence units (RLU) over a wide range of values and a ~30-fold brighter signal for NanoLuc luciferase (NLuc) over firefly luciferase (FLuc) (Extended Data Fig. 2c)

Summary

Introduction

Alternative splicing occurs in >90% of genes, and its impairment is associated with diseases such as spinal muscular atrophy and Parkinson’s disease. The minigene’s splice behaviour is read out by RT–qPCR or an embedded reporter gene This method can efficiently provide valuable insights into alternative splicing, it may not always reflect the physiological processes because partial intron/exon motifs may be overexpressed at unnatural levels, while essential regulatory sequences may be truncated. There is a trend towards increasing the size of the constructs by including large genomic fragments with multiple exons and full-length introns to better recapitulate splicing defects[16]. These ‘midigenes’ are cumbersome to assemble, usually requiring bacterial artificial chromosomes, and their sizes limit reasonable efficiencies in plasmid transfections. It is improbable that the regulatory machinery can be faithfully recapitulated outside of the precise three-dimensional genomic architecture at the endogenous sites

Methods

Results

Conclusion