RNA-Seq reveals widespread intronic polyadenylation diversifies immune cell transcriptomes

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3’UTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. Researchers from Memorial Sloan Kettering Cancer Center analyzed 46 3′-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein-protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide.

Widespread intronic polyadenylation in immune cells

a Schematic representation of full-length and IpA isoform of IGHM expressed in mature B cells and plasma cells (PC). b The 3ʹ-seq (tags per million (TPM)) and RNA-seq (read coverage) tracks showing expression of the IpA and full-length mRNA isoforms of IGHM (ENSG00000211899), encoding the immunoglobulin mu heavy chain, IgM. c The 3ʹ-seq tracks showing IpA isoform expression for two genes across human tissues and immune cell types. d RNA-seq coverage of intronic regions flanking IpA sites. A GLM-based test is used to validate the IpA isoforms. An isoform is considered validated if there is a significant difference (FDR-adjusted P < 0.1) in read counts in windows located up- and downstream of the putative IpA site. e The fraction of IpA isoforms validated by read evidence from independent data sets is shown for annotated and unannotated IpA isoforms. IpA isoforms present in RefSeq, UCSC genes, and Ensembl databases are considered to be annotated. f The fraction of expressed genes that generate IpA isoforms is shown for each cell type. g Expression levels (log₂ TPM) for full-length mRNAs and IpA isoforms are shown as boxplots for in PCs, blood NB, and T cells. IpA isoforms are robustly expressed as full-length mRNA expression is 4.53, 5.15, and 5.11, respectively, compared to 3.71, 3.83, and 3.71 for IpA isoforms. h Tissue-specific expression of IpA isoforms. Each row represents a gene. Dark blue, IpA isoform is expressed (≥5 TPM); light blue, IpA isoform is not expressed, but full-length mRNA is expressed (≥5.5 TPM); and white, gene is not expressed