Single-molecule, full-length transcript isoform sequencing reveals disease-associated RNA isoforms in cardiomyocytes

• Published in: Nature communications 2021-07, Vol.12 (1), p.4203-9, Article 4203
• Main Authors: Zhu, Chenchen, Wu, Jingyan, Sun, Han, Briganti, Francesca, Meder, Benjamin, Wei, Wu, Steinmetz, Lars M.
• Format: Article
• Language: English
• Subjects: 38; 38/91; 631/208/1792; 631/208/199; 692/699/75/74; Alternative Splicing; Annotations; Cardiomyocytes; Cardiomyopathy; Cardiomyopathy, Dilated - genetics; Cardiomyopathy, Dilated - pathology; Cardiovascular diseases; Cell Differentiation - genetics; Cell Line; Comparative analysis; Complexity; Computer applications; CRISPR-Cas Systems - genetics; Dilated cardiomyopathy; Eukaryotes; Exons; Feasibility Studies; Gene Editing; Gene sequencing; Genomes; Humanities and Social Sciences; Humans; Induced Pluripotent Stem Cells; Isoforms; Mitochondrial Proteins - genetics; Molecular Sequence Annotation; multidisciplinary; Muscle Proteins - genetics; Mutation; Myocytes, Cardiac - pathology; Ribonucleic acid; RNA; RNA Isoforms - genetics; RNA, Guide, CRISPR-Cas Systems; RNA-Binding Proteins - genetics; RNA-Seq - methods; Science; Science (multidisciplinary); Splicing; Transcription; Transcriptomes
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-021-24484-z

Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 ( RBM20 ) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons. Alternative splicing generates RNA isoforms that contribute to phenotypic diversity. Here the authors perform single-molecule full-length RNA sequencing to identify disease-associated variant transcript isoforms.