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

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.

Intron-encoded cistronic transcripts for minimally invasive monitoring of coding and non-coding RNAs

“…Please note that the general cloning procedures for generating INSPECT reporter cell lines are similar to our protocols published for EXSISERS 53 . A detailed exemplary protocol can be found on Protocol Exchange 54 .…”

Disease modification in Parkinsonism: obstacles and ways forward

“…Since, tau aggregates in PSP are mainly consistent of 4-repeat tau, another therapeutical approach would be to modify the splicing process of MAPT , to shift the presence of tau-protein from 4-repeat- to 3-repeat isoforms. Recently, a reporter system was developed that allows screening for specific splicing modifiers of MAPT (Truong et al 2021 ). Thus, in the future, pharmacological interventions that modify MAPT-splicing might be discovered and eventually used in clinical care.…”

Does the Anti‐Tau Strategy in Progressive Supranuclear Palsy Need to Be Reconsidered? No

“…In the preclinical setting, there are upcoming tau‐targeting opportunities by stimulating cellular defense mechanisms against misfolded tau, 25 by reducing tau expression, 26 or by altering tau splicing and thereby isoform expression. 27 A more comprehensive presentation of tau‐targeting opportunities is presented elsewhere. 5 …”

“…Also silencing of tau expression by intrathecal injection of antisense oligonucleotides, proven efficacious in tauopathy mouse models, 24 is currently being evaluated in a phase 1 study in PSP (NCT04539041). In the preclinical setting, there are upcoming tau‐targeting opportunities by stimulating cellular defense mechanisms against misfolded tau, 25 by reducing tau expression, 26 or by altering tau splicing and thereby isoform expression 27 . A more comprehensive presentation of tau‐targeting opportunities is presented elsewhere 5 …”

“…In the preclinical setting, there are upcoming tau-targeting opportunities by stimulating cellular defense mechanisms against misfolded tau, 25 by reducing tau expression, 26 or by altering tau splicing and thereby isoform expression. 27 A more comprehensive presentation of tau-targeting opportunities is presented elsewhere. 5 Of course, our perspective has to remain wide open so as not to miss any relevant therapeutic opportunities, including the relevance of tau loss-of-function mechanisms, 28 cell types other than neurons (eg, astrocytes), 29,30 neuroinflammation, 31 and energy failure.…”