The IpA isoform of is translated into a truncated protein (1C174 amino acids) that retains only its N-terminal domain (Fig

The IpA isoform of is translated into a truncated protein (1C174 amino acids) that retains only its N-terminal domain (Fig.?6e). domains or alter the binding specificity of proteins Vialinin A with DNA-binding or proteinCprotein 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. Recognition of intronic polyadenylation (IpA) signals can lead to expression of truncated proteins lacking C terminal domains. Analysis of 3? -seq and RNA-seq shows that IpA is widespread in circulating immune cells, while multiple myeloma cells show loss of IpA isoforms that are normally expressed in plasma cells, impacting key genes in the disease. Introduction Alternative cleavage and polyadenylation (ApA) is generally viewed as the selection of ApA signals in the 3??untranslated region (3?UTR), leading to the expression of different 3?UTR isoforms that code for Vialinin A the same protein. Recent computational analyses of 3?-end sequencing data have characterized the nature and extent of ApA in mammalian 3?UTRs1C7. For example, analysis of a human ApA tissue atlas established that half of human genes express multiple 3?UTRs, enabling tissue-specific post-transcriptional regulation of ubiquitously expressed genes1. However, ApA events can also occur in introns rather than 3?UTRs, generating either non-coding transcripts or transcripts with truncated coding regions that lead to loss of C-terminal domains in the protein product. The most famous example of cell type-specific usage of an intronic polyadenylation (IpA) signal occurs in the immunoglobulin M heavy chain (expressed in mature B cells and plasma cells (PC). b The 3?-seq (tags per million (TPM)) and RNA-seq (read coverage) tracks showing expression of the IpA and full-length mRNA isoforms of (ENSG00000211899), encoding the immunoglobulin mu heavy chain, IgM. The full-length isoform is expressed in NB from blood and lymphoid tissue and includes two exons encoding the C-terminal transmembrane domain of membrane-bound IgM. The IpA isoform is expressed in PCs obtained from bone marrow. It lacks the transmembrane domain which leads to expression of soluble IgM. c The 3?-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 is highly expressed in PCs while the full-length transcript, encoding membrane-bound IgM, is the dominant isoform in NB cells (Fig.?1b). Analysis of 3?-seq also revealed putative IpA isoforms, including in the locus of is well expressed in skeletal muscle and immune cells, and indeed is the only isoform expressed in PC, blood NB, and T cells; Fli1 these three cell types are also the only ones to express the IpA isoform of (Fig.?1d). Formally, we can test if RNA-seq read counts are significantly higher in intronic windows chosen upstream compared to downstream of IpA events (see Methods)17. We confirmed significantly differential coverage at 29% (has six ankyrin domains, while the IpA-generated truncated protein retains four of them. Similarly, the full-length protein of the transcription factor PATZ1 has Vialinin A seven zinc-finger domains, while different IpA isoforms are predicted to encode either four or five zinc-fingers (Fig.?4d). The partial loss of DNA-binding domains potentially changes DNA-binding specificity and therefore the set of regulated target genes. Similarly, the partial loss of PPIs can change the binding affinity to protein interaction partners. Open in a separate window Fig. 4 IpA isoforms diversify the proteome. a Genes that express IpA isoforms encode significantly larger proteins compared to genes that only express full-length mRNAs (one-sided Wilcoxon rank-sum test, < 10-5;?***< 10-10. b Genes that express IpA isoforms encode proteins with significantly more protein domains than genes that only express full-length mRNAs (one-sided Wilcoxon rank-sum test, nloses all zinc-finger domains encoded by the full-length transcript, potentially leading to expression of a truncated protein isoform with no known domain. While the IpA isoform is the dominant isoform in PC, it is almost completely lost in MM group 1 patients, which instead aberrantly express the full-length transcript. The gene encodes a component of the DDB1-CUL4A-ROC1 E3 ubiquitin ligase complex involved in Cereblon (CRBN)-mediated degradation of IKZF1 by lenalidomide35. The IpA isoform of is translated into a truncated protein (1C174 amino acids) that retains only its N-terminal domain (Fig.?6e). is overexpressed/amplified in other cancers37C40 and restricts cellular DNA damage repair41, and sensitivity to thalidomide correlates with CUL4A expression in.