Isoform Repertoire Research Articles

Cochlear transcript diversity and its role in auditory functions implied by an otoferlin short isoform

Isoforms of a gene may contribute to diverse biological functions. In the cochlea, the repertoire of alternative isoforms remains unexplored. We integrated single-cell short-read and long-read RNA sequencing techniques and identified 236,012 transcripts, 126,612 of which were unannotated in the GENCODE database. Then we analyzed and verified the unannotated transcripts using RNA-seq, RT-PCR, Sanger sequencing, and MS-based proteomics approaches. To illustrate the importance of identifying spliced isoforms, we investigated otoferlin, a key protein involved in synaptic transmission in inner hair cells (IHCs). Upon deletion of the canonical otoferlin isoform, the identified short isoform is able to support normal hearing thresholds but with reduced sustained exocytosis of IHCs, and further revealed otoferlin functions in endocytic membrane retrieval that was not well-addressed previously. Furthermore, we found that otoferlin isoforms are associated with IHC functions and auditory phenotypes. This work expands our mechanistic understanding of auditory functions at the level of isoform resolution.

A Putative TRAPα Protein of Microsporidia Nosema bombycis Exhibits Non-Canonical Alternative Polyadenylation in Transcripts.

Microsporidia are obligate intracellular eukaryotic parasites that have significantly reduced genomes and that have lost most of their introns. In the current study, we characterized a gene in microsporidia Nosema bombycis, annotated as TRAPα (HNbTRAPα). The homologous of TRAPα are a functional component of ER translocon and facilitates the initiation of protein translocation in a substrate-specific manner, which is conserved in animals but absent from most fungi. The coding sequence of HNbTRAPα consists of 2226 nucleotides, longer than the majority of homologs in microsporidia. A 3' RACE analysis indicated that there were two mRNA isoforms resulting from non-canonical alternative polyadenylation (APA), and the polyadenylate tail was synthesized after the C951 or C1167 nucleotide, respectively. Indirect immunofluorescence analysis showed two different localization characteristics of HNbTRAPα, which are mainly located around the nuclear throughout the proliferation stage and co-localized with the nuclear in mature spores. This study demonstrated that the post-transcriptional regulation mechanism exists in Microsporidia and expands the mRNA isoform repertoire.

Isoform requirement of clustered protocadherin for preventing neuronal apoptosis and neonatal lethality

Clustered protocadherin is a family of cell-surface recognition molecules implicated in neuronal connectivity that has a diverse isoform repertoire and homophilic binding specificity. Mice have 58 isoforms, encoded by Pcdhα, β, and γ gene clusters, and mutant mice lacking all isoforms died after birth, displaying massive neuronal apoptosis and synapse loss. The current hypothesis is that the three specific γC-type isoforms, especially γC4, are essential for the phenotype, raising the question about the necessity of isoform diversity. We generated TC mutant mice that expressed the three γC-type isoforms but lacked all the other 55 isoforms. The TC mutants died immediately after birth, showing massive neuronal death, and γC3 or γC4 expression did not prevent apoptosis. Restoring the α- and β-clusters with the three γC alleles rescued the phenotype, suggesting that along with the three γC-type isoforms, other isoforms are also required for the survival of neurons and individual mice.

Exploring Splicing Factor Aberrations As a Therapeutic Target in Mature B-Cell Malignancies

Objectives Cancers originating from mature B cells at various stages of terminal differentiation represent a diverse set of cancers. There are numerous pathological subtypes, each comprising genetically and clinically heterogeneous malignancies with distinct biology and clinical outcomes. We have previously identified the recurrence of mutations in splicing factor HNRNPH1 in mantle cell lymphoma (MCL), which were associated with inferior outcomes. Based on growing evidence for the role of splicing factors in mature B cell neoplasms, we sought to extend our characterization of splicing factor aberrations across additional B cell malignancies. We hypothesize that regulation of alternative splicing is essential for B cell differentiation and maturation, and aberrations of splicing factors in B cell malignancies are related to these normal patterns of alternative splicing. Ultimately, we are pursuing alternative splicing as a potential novel therapeutic vulnerability common among mature B cell neoplasms. Methods We leveraged whole genome, exome, and RNA sequencing data from 2999 tumors assembled from controlled-access repositories and ongoing projects at BC Cancer as part of our Genomic Analysis of Mature B-cell Lymphomas (GAMBL) project. RNA-seq data was aligned with STAR and splicing events were quantified with RMATS. Novel splicing events were quantified following de novo transcriptome assembly with StringTie. Simple somatic mutations were detected using a workflow comprised of four algorithms to identify high-confidence variants. We used additional RNA-seq data from distinct B-cell maturation states (naïve, centroblast, centrocyte, memory, bone marrow plasma, and tonsillar plasma cells) to identify annotated and novel splicing events which are differentially regulated throughout B cell maturation. Annotated and novel events were subsequently used to query alternative splicing in a subset of tumor samples using a combination of Illumina-based and Oxford Nanopore sequencing. Results More than 30% of patient samples harboured somatic mutations in at least one of 67 queried splicing factors. HNRNPU, PCBP1, SF3B1, DAZAP1, and HNRNPH1 were most commonly mutated, 13% of cases (Figure 1A). Although only reported in MCL thus far, we noted an abundance of HNRNPH1 mutations in DLBCL showing the same pattern of non-coding mutations, most commonly in the activated B cell subgroup. In non-malignant samples, we identified 129 known and 267 novel splicing events which display differential regulation during B cell maturation and warrant further investigation within subtypes of B cell malignancies (Figure 1B). Based on this observation, we hypothesize that the RNA isoform repertoire may vary across B cell differentiation states and its perturbation may contribute to oncogenesis in a context-dependent manner. Furthermore, the importance of alternative splicing during B cell maturation implies that perturbation of splicing in B cell malignancies could play a role in pathogenesis. We previously demonstrated that non-coding mutations in HNRNPH1 contribute to overexpression of the hnRNP H protein and are associated with poor patient outcomes in MCL. Based on the predominance of these mutations in DLBCL, we sought to evaluate hnRNP H overexpression as a potential therapeutic target. We identified a putative exonic splicing enhancer (ESE) with minimal secondary structure in the essential exon of HNRNPH1 using ESEfinder and designed locked nucleic acid antisense oligos (ASOs) complementary to the ESE. Using ddPCR, we showed that the ESE ASO can effectively decrease the proportion of the productive HNRNPH1 splice variant at nanomolar concentrations in HEK cells; however, a splice site ASO does not display the same efficacy. Conclusions We have identified a growing number of splicing factors in mature B cell malignancies as recurrently mutated, indicating that deregulation of the RNA isoform repertoire in B cells can contribute to oncogenesis. Characterizing the landscape of alternative splicing in normal and malignant B cells will allow us to identify opportunities to exploit this reliance in the development of novel therapeutic strategies. Our data highlight antisense oligonucleotides as a promising therapeutic avenue for targeting splicing aberrations caused by common mutations in B cell malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Transcriptome innovations in primates revealed by single-molecule long-read sequencing.

Transcriptomic diversity greatly contributes to the fundamentals of disease, lineage-specific biology, and environmental adaptation. However, much of the actual isoform repertoire contributing to shaping primate evolution remains unknown. Here, we combined deep long- and short-read sequencing complemented with mass spectrometry proteomics in a panel of lymphoblastoid cell lines (LCLs) from human, three other great apes, and rhesus macaque, producing the largest full-length isoform catalog in primates to date. Around half of the captured isoforms are not annotated in their reference genomes, significantly expanding the gene models in primates. Furthermore, our comparative analyses unveil hundreds of transcriptomic innovations and isoform usage changes related to immune function and immunological disorders. The confluence of these evolutionary innovations with signals of positive selection and their limited impact in the proteome points to changes in alternative splicing in genes involved in immune response as an important target of recent regulatory divergence in primates.

P508. Assessing Isoform Diversity in the Context of Psychiatric Disorders

The genetic architecture of schizophrenia is dominated by common variants of small effects, similar to most complex human traits. The majority of the schizophrenia GWAS SNPs reside in non-coding regions where they could act through a variety of regulatory mechanisms, such as alternative splicing to generate schizophrenia-associated isoforms. The brain expresses the most diverse repertoire of isoforms, yet our understanding of brain-specific isoform expression and the mechanisms of their regulation by RNA binding proteins (RBPs) are incomplete.

A TLR/CD44 axis regulates T cell trafficking in experimental and human multiple sclerosis

SummaryIn the pathogenesis of autoimmune disorders, the modulation of leukocytes′ trafficking plays a central role, still poorly understood. Here, we focused on the effect of TLR2 ligands in trafficking of T helper cells through reshuffling of CD44 isoforms repertoire. Concurrently, strain background and TLR2 haplotype affected Wnt/β-catenin signaling pathway and expression of splicing factors. During EAE, mCD44v9-v10 was specifically enriched in the forebrain and showed an increased ability to bind stably to osteopontin. Similarly, we observed that hCD44v7 was highly enriched in cells of cerebrospinal fluid from MS patients with active lesions. Moreover, TLRs engagement modulated the composition of CD44 variants also in human T helper cells, supporting the hypothesis that pathogens or commensals, through TLRs, in turn modulate the repertoire of CD44 isoforms, thereby controlling the distribution of lesions in the CNS. The interference with this mechanism(s) represents a potential tool for prevention and treatment of autoimmune relapses and exacerbations.

RBFOX splicing factors contribute to a broad but selective recapitulation of peripheral tissue splicing patterns in the thymus.

Thymic epithelial cells (TEC) control the selection of a T cell repertoire reactive to pathogens but tolerant of self. This process is known to involve the promiscuous expression of virtually the entire protein-coding gene repertoire, but the extent to which TEC recapitulate peripheral isoforms, and the mechanisms by which they do so, remain largely unknown. We performed the first assembly-based transcriptomic census of transcript structures and splicing factor (SF) expression in mouse medullary TEC (mTEC) and 21 peripheral tissues. Mature mTEC expressed 60.1% of all protein-coding transcripts, more than was detected in any of the peripheral tissues. However, for genes with tissue-restricted expression, mTEC produced fewer isoforms than did the relevant peripheral tissues. Analysis of exon inclusion revealed an absence of brain-specific microexons in mTEC. We did not find unusual numbers of novel transcripts in TEC, and we show that Aire, the facilitator of promiscuous gene expression, promotes the generation of long “classical” transcripts (with 5′ and 3′ UTRs) but has only a limited impact on alternative splicing in mTEC. Comprehensive assessment of SF expression in mTEC identified a small set of nonpromiscuously expressed SF genes, among which we confirmed RBFOX to be present with AIRE in mTEC nuclei. Using a conditional loss-of-function approach, we show that Rbfox2 promotes mTEC development and regulates the alternative splicing of promiscuously expressed genes. These data indicate that TEC recommission a small number of peripheral SFs, including members of the RBFOX family, to generate a broad but selective representation of the peripheral splice isoform repertoire.

Long read transcript profiling of ion channel splice isoforms.

Alternative splicing of RNA transcripts allows a single gene to generate multiple products and is a key means of generating functionally diverse voltage-gated ion channels. Splicing can be regulated according to cell type, cell state, and stage of development to produce a bespoke complement of protein isoforms. Characterizing the identities of full-length transcript isoforms is essential in order to fully understand a gene's expression and function. However, the repertoire of transcript isoforms is not well characterized for most genes. Long read nanopore sequencing allows full-length isoforms to be sequenced, therefore identifying full-length transcripts. Using this approach, we recently discovered that the human CACNA1C gene gives rise to a far greater repertoire of splice isoforms than previously appreciated. Here we provide a detailed overview of the technical approach we used to achieve this. The method described in this chapter combines long read nanopore sequencing with PCR targeting to selectively sequence transcripts of a specific gene of interest.

Poison Exon Splicing Regulates a Coordinated Network of SR Protein Expression during Differentiation and Tumorigenesis

The RNA isoform repertoire is regulated by splicing factor (SF) expression, and alterations in SF levels are associated with disease. SFs contain ultraconserved poison exon (PE) sequences that exhibit greater identity across species than nearby coding exons, but their physiological role and molecular regulation is incompletely understood. We show that PEs in serine-arginine-rich (SR) proteins, a family of 14 essential SFs, are differentially spliced during induced pluripotent stem cell (iPSC) differentiation and in tumors versus normal tissues. We uncover an extensive cross-regulatory network of SR proteins controlling their expression via alternative splicing coupled to nonsense-mediated decay. We define sequences that regulate PE inclusion and protein expression of the oncogenic SF TRA2β using an RNA-targeting CRISPR screen. We demonstrate location dependency of RS domain activity on regulation of TRA2β-PE using CRISPR artificial SFs. Finally, we develop splice-switching antisense oligonucleotides to reverse the increased skipping of TRA2β-PE detected in breast tumors, altering breast cancer cell viability, proliferation, and migration.

Isoform repertoire of Blood mononuclear cells from SLE patients

Abstract Systemic Lupus Erythematosus (SLE) is characterized by numerous immunological alterations, including increased expression of immune activators, such as IFNa, along with alterations in cell composition, such as expansion of plasma cells that produce autoantibodies. Alternative Splicing (AS) is a biological process that regulates post-transcriptional alterations of the RNA. Pathogenic AS events have been associated to several diseases. Furthermore, the description of SLE autoantibodies that target components of the spliceosome such as anti-Sm/RNP, anti-Ro/La, and anti-dsDNA has led us to hypothesize that SLE is associated to abnormal AS events. RNA-seq uses standard Short-Read technologies which involve RNA or cDNA fragmentation for the generation of 150 bp sequencing libraries. Since most mammalian mRNAs are 1–2 kb long, using RNA-seq to identify novel alternatively spliced isoforms is extremely challenging. However, emerging Long-Read sequencing technologies allow full-length mRNA sequencing, de novo transcriptome assembly, and isoform profiling. Our lab has built a computational pipeline that combines Long-Read sequencing with RNA-seq data to identify and quantify novel transcripts in peripheral blood mononuclear cells (PBMCs) from SLE patients compared to healthy controls. From our computational analysis, we have observed that SLE patients display differential exon-inclusion and exclusion events in transcripts coding for immune activating and regulatory proteins, respectively. We conclude that SLE might be associated with pathogenic AS events that might place the immune balance under double jeopardy, through activation of immune activating receptors and inhibition of immune inhibiting receptors.

Unraveling Mechanisms of Patient-Specific NRXN1 Mutations in Neuropsychiatric Diseases Using Human Induced Pluripotent Stem Cells.

Rare heterozygous deletions in the neurexin 1 (NRXN1) gene robustly increase an individual's risk of developing neurological and psychiatric disorders. However, the molecular bases by which different mutations result in different clinical presentations, with variable penetrance, are unknown. To better understand the molecular and cellular consequences of heterozygous NRXN1 mutations, Flaherty and colleagues studied how patient mutations influence the NRXN1 isoform repertoire and neuronal phenotypes using induced pluripotent stem (iPS) cells. Advancing from disease association to mechanistic insights, the authors provide insight into how patient mutations might impinge on neuronal function. This research highlights the value of iPS cells for elucidating otherwise elusive links between molecular and neuronal function. In addition, they provide further evidence of the importance of alternative splicing in the pathophysiology of neuropsychiatric diseases.

Circular RNAs add diversity to androgen receptor isoform repertoire in castration-resistant prostate cancer.

Deregulated expression of circular RNAs (circRNAs) is associated with various human diseases, including many types of cancer. Despite their growing links to cancer, there has been limited characterization of circRNAs in metastatic castration-resistant prostate cancer, the major cause of prostate cancer mortality. Here, through the analysis of an exome-capture RNA-seq dataset from 47 metastatic castration-resistant prostate cancer samples and ribodepletion and RNase R RNA-sequencing of patient-derived xenografts (PDXs) and cell models, we identified 13 circRNAs generated from the key prostate cancer driver gene-androgen receptor (AR). We validated and characterized the top four most abundant, clinically relevant AR circRNAs. Expression of these AR circRNAs was upregulated during castration-resistant progression of PDXs. The upregulation was not due to global increase of circRNA formation in these tumors. Instead, the levels of AR circRNAs correlated strongly with that of the linear AR transcripts (both AR and AR-variants) in clinical samples and PDXs, indicating a transcriptional mechanism of regulation. In cultured cells, androgen suppressed the expression of these AR circRNAs and the linear AR transcripts, and the suppression was attenuated by an antiandrogen. Using nuclear/cytoplasmic fractionation and RNA in-situ hybridization assays, we demonstrated predominant cytoplasmic localization of these AR circRNAs, indicating likely cytoplasmic functions. Overall, this is the first comprehensive characterization of circRNAs arising from the AR gene. With greater resistance to exoribonuclease compared to the linear AR transcripts and detectability of AR circRNAs in patient plasma, these AR circRNAs may serve as surrogate circulating markers for AR/AR-variant expression and castration-resistant prostate cancer progression.

HnRNPC regulates cancer-specific alternative cleavage and polyadenylation profiles.

Alternative cleavage and polyadenylation (APA) can occur at more than half of all human genes, greatly enhancing the cellular repertoire of mRNA isoforms. As these isoforms can have altered stability, localisation and coding potential, deregulation of APA can disrupt gene expression and this has been linked to many diseases including cancer progression. How APA generates cancer-specific isoform profiles and what their physiological consequences are, however, is largely unclear. Here we use a subcellular fractionation approach to determine the nuclear and cytoplasmic APA profiles of successive stages of colon cancer using a cell line-based model. Using this approach, we show that during cancer progression specific APA profiles are established. We identify that overexpression of hnRNPC has a critical role in the establishment of APA profiles characteristic for metastatic colon cancer cells, by regulating poly(A) site selection in a subset of genes that have been implicated in cancer progression including MTHFD1L.

Defining the genetic and evolutionary architecture of alternative splicing in response to infection

Host and environmental factors contribute to variation in human immune responses, yet the genetic and evolutionary drivers of alternative splicing in response to infection remain largely uncharacterised. Leveraging 970 RNA-sequencing profiles of resting and stimulated monocytes from 200 individuals of African- and European-descent, we show that immune activation elicits a marked remodelling of the isoform repertoire, while increasing the levels of erroneous splicing. We identify 1,464 loci associated with variation in isoform usage (sQTLs), 9% of them being stimulation-specific, which are enriched in disease-related loci. Furthermore, we detect a longstanding increased plasticity of immune gene splicing, and show that positive selection and Neanderthal introgression have both contributed to diversify the splicing landscape of human populations. Together, these findings suggest that differential isoform usage has been an important substrate of innovation in the long-term evolution of immune responses and a more recent vehicle of population local adaptation.

Revisiting Dscam diversity: lessons from clustered protocadherins.

The complexity of neuronal wiring relies on the extraordinary recognition diversity of cell surface molecules. Drosophila Dscam1 and vertebrate clustered protocadherins (Pcdhs) are two classic examples of the striking diversity from a complex genomic locus, wherein the former encodes more than 10,000 distinct isoforms via alternative splicing, while the latter employs alternative promoters to attain isoform diversity. These structurally unrelated families show remarkably striking molecular parallels and even similar functions. Recent studies revealed a novel Dscam gene family with tandemly arrayed 5' cassettes in Chelicerata (e.g., the scorpion Mesobuthus martensii and the tick Ixodes scapularis), similar to vertebrate clustered Pcdhs. Likewise, octopus shows a more remarkable expansion of the Pcdh isoform repertoire than human. These discoveries of Dscam and Pcdh diversification reshape the evolutionary landscape of recognition molecule diversity and provide a greater understanding of convergent molecular strategies for isoform diversity. This article reviews new insights into the evolution, regulatory mechanisms, and functions of Dscam and Pcdh isoform diversity. In particular, the convergence of clustered Dscams and Pcdhs is highlighted.

Abstract 4440: Differential regulation of p63 isoforms by alternative polyadenylation

Abstract p63, a transcription factor belonging to the p53 family, plays vital roles in epidermal development and tumorigenesis. p63 expresses from two different promoters generating two classes of proteins, TAp63 and ΔNp63 that differ in their N-terminus. Moreover, alternative splicing of the TA and ΔN isoforms in the C-terminus creates additional isoforms (α-ϵ); thus generating five TA and five ΔN p63 isoforms. The factors that give rise to this repertoire of p63 isoforms are not very well understood. Alternative polyadenylation (APA) can generate multiple isoforms of a gene with alternative 3' ends based on the polyadenylation site selection during cleavage and polyadenylation of mRNA. APA can give rise to isoforms that differ in the coding region (CDS APA) or that have varying lengths of 3' UTR (3' UTR APA). Recently, PABPN1 protein that binds the 3' poly(A) tail of mRNAs has emerged as a key regulator of APA. Here, we show that PABPN1 differentially regulates p63α and p63γ isoforms by alternative polyadenylation. Specifically, knockdown of PABPN1 increases the levels of p63γ mRNA as well as protein by regulating CDS APA of p63 gene. Moreover, knockdown of PABPN1 also generates p63α mRNAs with varying 3' UTR lengths. Although the mRNA levels of p63α remain unchanged upon PABPN1 KD, p63α protein is significantly decreased, suggesting that PABPN1 also regulates p63α protein translation. Interestingly, we also show that PABPN1 is a p63α target and that p63 transcriptionally regulates PABPN1 by binding to its promoter forming a negative feedback loop. Taken together, our study identifies PABPN1 as a novel regulator of various p63 isoforms by APA and that p63 in turn regulates PABPN1 transcriptionally. Citation Format: Shakur Mohibi, Jin Zhang, Xinbin Chen. Differential regulation of p63 isoforms by alternative polyadenylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4440.

A chelicerate-specific burst of nonclassical Dscam diversity

BackgroundThe immunoglobulin (Ig) superfamily receptor Down syndrome cell adhesion molecule (Dscam) gene can generate tens of thousands of isoforms via alternative splicing, which is essential for both nervous and immune systems in insects. However, further information is required to develop a comprehensive view of Dscam diversification across the broad spectrum of Chelicerata clades, a basal branch of arthropods and the second largest group of terrestrial animals.ResultsIn this study, a genome-wide comprehensive analysis of Dscam genes across Chelicerata species revealed a burst of nonclassical Dscams, categorised into four types—mDscam, sDscamα, sDscamβ, and sDscamγ—based on their size and structure. Although the mDscam gene class includes the highest number of Dscam genes, the sDscam genes utilise alternative promoters to expand protein diversity. Furthermore, we indicated that the 5′ cassette duplicate is inversely correlated with the sDscam gene duplicate. We showed differential and sDscam- biased expression of nonclassical Dscam isoforms. Thus, the Dscam isoform repertoire across Chelicerata is entirely dominated by the number and expression levels of nonclassical Dscams. Taken together, these data show that Chelicerata evolved a large conserved and lineage-specific repertoire of nonclassical Dscams.ConclusionsThis study showed that arthropods have a large diversified Chelicerata-specific repertoire of nonclassical Dscam isoforms, which are structurally and mechanistically distinct from those of insects. These findings provide a global framework for the evolution of Dscam diversity in arthropods and offer mechanistic insights into the diversification of the clade-specific Ig superfamily repertoire.

Abstract 2442: Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System

Abstract Tremendous flexibility is maintained in the human proteome via alternative splicing, and cancer genomes often subvert this flexibility to promote survival. Identification and annotation of cancer-specific mRNA isoforms is critical to understanding how mutations in the genome affect the biology of cancer cells. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. In cancer proteomics studies, the identification of biomarkers from mass spectroscopy data is often limited by incomplete gene isoform expression information to support protein to transcript mapping. The Iso-Seq™ protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences needed to discover biomarkers for early detection and cancer stratification, to fully characterize gene fusion events, and to elucidate drug resistance mechanisms. Knowledge of the complete isoform repertoire is also key for accurate quantification of isoform abundance. As most transcripts range from 1 - 10 kb, fully intact RNA molecules can be sequenced using SMRT® Sequencing without requiring fragmentation or post-sequencing assembly. However, some cancer research applications have presented a challenge for the Iso-Seq protocol, due to the combination of limited sample input and the need to deeply sequence heterogenous samples. Here we report the optimization of the Iso-Seq library preparation protocol for the PacBio Sequel platform and its application to cancer cell lines and tumor samples. We demonstrate how loading enhancements on the higher-throughput Sequel instrument have decreased the need for size fractionation steps, reducing sample input requirements while simultaneously simplifying the sample preparation workflow and increasing the number of full-length transcripts per SMRT Cell. The results highlight the potential for broader application of the Iso-Seq method to more comprehensively characterize alternative splicing in cancer. Citation Format: Meredith Ashby, Ting Hon, Elizabeth Tseng, Aparna Vedula, Tyson A. Clark. Simplified sequencing of full-length isoforms in cancer on the PacBio Sequel System [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2442. doi:10.1158/1538-7445.AM2017-2442

Alternative polyadenylation expands the mRNA isoform repertoire of human CD46

Alternative polyadenylation is a prevalent mechanism regulating mammalian gene expression. While tandem 3′-Untranslated-Region (3′UTR) polyadenylation changes expression levels, Intronic PolyAdenylation generates shorter transcripts encoding truncated proteins. Intronic PolyAdenylation regulates 20% of genes and is especially common in receptor tyrosine-kinase transcripts, generating soluble repressors. Here we report that human CD46, encoding a TransMembrane repressor of complement and T-cell co-stimulator, expresses multiple isoforms by alternative polyadenylation. We provide evidence for polyadenylation at several introns by RT-PCR of 5′ intronic fragments, and by increase in such isoforms via functional U1 knockdown. We mapped various Intronic PolyAdenylation Sites by 3′ Rapid Amplification of cDNA Ends (3′RACE), which could generate soluble or membrane-bound but tail-less CD46. Intronic PolyAdenylation could add to the source of soluble CD46 isoforms in fluids and tissues, which increase in cancers and autoimmune syndromes. Furthermore, 3′RACE identified three PolyAdenylation Sites within the last intron and exon, whose transcripts with shortened 3′UTRs could support higher CD46 expression. Finally, 3′RACE revealed that the CD46 Pseudogene only expresses short transcripts by early polyadenylation in intron 2. Overall, we report a wide variety of CD46 mRNA isoforms which could generate new protein isoforms, adding to the diverse physiological and pathological roles of CD46.