Institute for Synthetic Biomedicine, Helmholtz Zentrum München, Oberschleißheim, Germany.
Department of Chemistry and TUM School of Medicine, Technical University of Munich, Munich, Germany.
Nat Cell Biol. 2021 Jun;23(6):652-663. doi: 10.1038/s41556-021-00678-x. Epub 2021 Jun 3.
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.
外显子特异性异构体的表达是一种基本的机制，它大大扩展了细胞的蛋白质组。然而，评估选择性剪接的传统方法要么消耗大量时间和精力，要么不能在蛋白质水平上进行纵向定量异构体表达。在这里，我们因此开发了一种外显子特异性异构体表达报告系统（EXSISERS），它通过高效的、内含子介导的蛋白质剪接，无痕地从新生多肽链中切除报告蛋白，从而非侵入性地报告内源性基因中外显子包含的异构体的翻译。我们应用 EXSISERS 来定量量化微管相关蛋白 tau (MAPT) 中与疾病相关的外显子 10 的包含情况，在患者来源的诱导多能干细胞中，并筛选基于 Cas13 的 RNA 靶向效应器的异构体特异性。我们还将细胞存活与参与维持胚胎干细胞多能性的 FOXP1 的外显子 18b 的包含相耦合，并证实 MBNL1 是外显子 18b 排除的主要因素。EXSISERS 能够以高灵敏度和细胞分辨率进行非破坏性和多模式监测外显子特异性异构体表达，并能够进行外显子特异性治疗干预的高通量筛选。
