Alternative Exon Research Articles

Dysregulation of alternative splicing is associated with the pathogenesis of ulcerative colitis

BackgroundAlthough numerous risk loci for ulcerative colitis (UC) have been identified in the human genome, the pathogenesis of UC remains unclear. Recently, multiple transcriptomic analyses have shown that aberrant gene expression in the colon tissues of UC patients is associated with disease progression. A pioneering study also demonstrated that altered post-transcriptional regulation is involved in the progression of UC. Here, we provide a genome-wide analysis of alternative splicing (AS) signatures in UC patients. We analyzed three datasets containing 74 tissue samples from UC patients and identified over 2000 significant AS events.ResultsSkipped exon and alternative first exon were the two most significantly altered AS events in UC patients. The immune response-related pathways were remarkably enriched in the UC-related AS events. Genes with significant AS events were more likely to be dysregulated at the expression level.ConclusionsWe present a genomic landscape of AS events in UC patients based on a combined analysis of two cohorts. Our results indicate that dysregulation of AS may have a pivotal role in determining the pathogenesis of UC. In addition, our study uncovers genes with potential therapeutic implications for UC treatment.

The Dysferlin Transcript Containing the Alternative Exon 40a is Essential for Myocyte Functions

Dysferlinopathies are a group of muscular dystrophies caused by recessive mutations in the DYSF gene encoding the dysferlin protein. Dysferlin is a transmembrane protein involved in several muscle functions like T-tubule maintenance and membrane repair. In 2009, a study showed the existence of fourteen dysferlin transcripts generated from alternative splicing. We were interested in dysferlin transcripts containing the exon 40a, and among them the transcript 11 which contains all the canonical exons and exon 40a. This alternative exon encodes a protein region that is cleaved by calpains during the muscle membrane repair mechanism. Firstly, we tested the impact of mutations in exon 40a on its cleavability by calpains. We showed that the peptide encoded by the exon 40a domain is resistant to mutations and that calpains cleaved dysferlin in the first part of DYSF exon 40a. To further explore the implication of this transcript in cell functions, we performed membrane repair, osmotic shock, and transferrin assay. Our results indicated that dysferlin transcript 11 is a key factor in the membrane repair process. Moreover, dysferlin transcript 11 participates in other cell functions such as membrane protection and vesicle trafficking. These results support the need to restore the dysferlin transcript containing the alternative exon 40a in patients affected with dysferlinopathy.

Parental Origin of Gsα Inactivation Differentially Affects Bone Remodeling in a Mouse Model of Albright Hereditary Osteodystrophy.

ABSTRACTAlbright hereditary osteodystrophy (AHO) is caused by heterozygous inactivation of GNAS, a complex locus that encodes the alpha‐stimulatory subunit of heterotrimeric G proteins (Gsα) in addition to NESP55 and XLαs due to alternative first exons. AHO skeletal manifestations include brachydactyly, brachymetacarpia, compromised adult stature, and subcutaneous ossifications. AHO patients with maternally‐inherited GNAS mutations develop pseudohypoparathyroidism type 1A (PHP1A) with resistance to multiple hormones that mediate their actions through G protein‐coupled receptors (GPCRs) requiring Gsα (eg, parathyroid hormone [PTH], thyroid‐stimulating hormone [TSH], growth hormone–releasing hormone [GHRH], calcitonin) and severe obesity. Paternally‐inherited GNAS mutations cause pseudopseudohypoparathyroidism (PPHP), in which patients have AHO skeletal features but do not develop hormonal resistance or marked obesity. These differences between PHP1A and PPHP are caused by tissue‐specific reduction of paternal Gsα expression. Previous reports in mice have shown loss of Gsα causes osteopenia due to impaired osteoblast number and function and suggest that AHO patients could display evidence of reduced bone mineral density (BMD). However, we previously demonstrated PHP1A patients display normal‐increased BMD measurements without any correlation to body mass index or serum PTH. Due to these observed differences between PHP1A and PPHP, we utilized our laboratory's AHO mouse model to address whether Gsα heterozygous inactivation differentially affects bone remodeling based on the parental inheritance of the mutation. We identified fundamental distinctions in bone remodeling between mice with paternally‐inherited (GnasE1+/−p) versus maternally‐inherited (GnasE1+/−m) mutations, and these findings were observed predominantly in female mice. Specifically, GnasE1+/−p mice exhibited reduced bone parameters due to impaired bone formation and enhanced bone resorption. GnasE1+/−m mice, however, displayed enhanced bone parameters due to both increased osteoblast activity and normal bone resorption. These in vivo distinctions in bone remodeling between GnasE1+/−p and GnasE1+/−m mice could potentially be related to changes in the bone microenvironment driven by calcitonin‐resistance within GnasE1+/−m osteoclasts. Further studies are warranted to assess how Gsα influences osteoblast–osteoclast coupling. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Transcriptome Based Classification Model and Co-Expression Networks Analysis Identify CXCR4alt Subtype and Blnkaf in B-Cell Precursor Acute Lymphoblastic Leukemia

BackgroundB-cell precursor acute lymphoblastic leukemia (B-ALL) is a genetically heterogeneous group of acute leukemia with stage-specific phenotypes and cytogenetic features. Although the research on the molecular profile of B-ALL benefits diagnosis and risk stratification, the idiographic leukemogenesis beyond the transcriptome remains unknown. Genomic lesions in B-ALL frequently involve genes belonging to transcription factors, such as TCF3, EBF1, PAX5, and IKZF1. The investigation of dysregulated transcriptional networks behind various B-ALL subtypes may help unravel the specific process of leukemogenesis.MethodsA random forest model was trained on a well-defined molecular subtype B-ALL cohort (n = 504) to improve the molecular classification. The subtype-specific transcriptional network was constructed by weighted correlation network analysis (WGCNA) once the B-ALL subtypes were genetically determined by the random forest model. Additionally, alternative splicing analysis from RNA-seq was emphasized since aberrant splicing events could lead to abnormalities in transcription factors or tumor suppressor genes.ResultsThe random forest model performs well for the classification of most B-ALL subtypes (Figure 1A). It also benefits the classification of Ph-like B-ALL, which displays a gene expression profile similar to BCR-ABL1 B-ALL, as it achieves 100% accuracy on well-known Ph-like cases characterized by ABL-class gene fusions, PAX5-JAK2, EBF1-PDGFRB, and IGH-EPOR.We successfully separated a candidate molecular subtype characterized by CXCR4 alteration (CXCR4alt) for the first time, through our novel classification model (Figure 1B). This newly identified CXCR4alt subtype accounts for 2% of B-ALL cases (11/504), characterized by CXCR4 C-terminal mutation R334X or FLNA overexpression. Both C-terminal mutation and upregulated FLNA contribute to delayed CXCR4 receptor internalization, enhanced CXCL12-CXCR4 signaling, and then continuously activates the downstream MAPK pathway. It is further supported by the high expression of the two oncogenic MAPK signaling pathway genes KIAA1549 and KIAA1549L from the co-expression network of CXCR4alt in these cases.Transcriptional co-expression networks constructed by WGCNA and network hub genes for most B-ALL subtypes also help to elucidate the mechanism of leukemogenesis (Figure 2). We identified an alternative first exon of BLNK (BLNKaf) that leads to loss of function as a shared event in specific subtypes, such as BCR-ABL1, BCR-ABL1-like, and PAX5alt; while in pre-BCR signaling positive subtypes, such as TCF3-PBX1 and MEF2D-r, only express normal BLNK transcripts.DiscussionBy comprehensive transcriptome-based classification model and co-expression networks analysis, we identified a novel defined CXCR4alt subtype with an incidence of 2% in B-ALL. We also observed that BLNKaf might supply a practical marker for monitoring pre-BCR signaling. Our report emphasizes the role of transcriptome-based machine learning and WGCNA in mining the molecular mechanism of B-ALL. The molecular pathogenesis and clinical significance of these newly identified molecular subtypes and molecular abnormalities are worthy of further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transcriptional Profile Investigation Identified Aberrant CD34 Isoforms and Apela As a Hub Gene in IGH-DUX4 Subtype B-Cell Precursor Acute Lymphoblastic Leukemia

BackgroundCD34, a transmembrane sialoglycoprotein, is expressed in hematopoietic stem and progenitor cells, endothelial cells, and bone marrow stromal cells. Therefore, in the clinical diagnosis and classification of leukemia, high expression of CD34 is customized as a marker for the immature phenotype. Although alternative splicing is a common oncogenic mechanism in various cancers, abnormal splicing of CD34 has not been revealed in hematological malignancies.MethodsHere, we investigated the transcriptional profile of CD34, including expression level and alternative splicing, by RNA sequencing (RNA-seq) analysis from hematological malignancies and normal bone marrow samples. The raw sequencing reads were aligned to human reference genome hg38 using HISAT2, followed by featureCounts quantification, and co-expressed and differentially expressed genes (DEGs) were detected by WGCNA and DESeq2, respectively. Alternative splicing events were calculated by rMATS, and further validated by reverse transcriptase PCR (RT-PCR).ResultsThe transcriptome characteristics of hematological malignancies, including AML, B-ALL, T-ALL, and MPAL, were elaborately investigated. We found that two accompanying novel in-frame splicing isoforms of CD34 were exclusively detected in B-ALL. Furthermore, we focused on B-ALL (n = 504) to systematically explore the transcriptional profile of CD34. The two novel splicing isoforms share a common first exon at the 5' untranslated region of CD34 (Figure 1B), suggesting an alternative promoter that mediated the splicing.We further observe that the novel aberrant CD34 isoforms are mainly accompanied by IGH-DUX4 gene fusion, which has been reported characterized by high expression of CD34 and intragenic ERG deletion. All IGH-DUX4 B-ALL cases (n = 20) in our cohort were positive with the novel aberrant CD34 isoforms and validated by RT-PCR (Figure 1A). We also validated the negative result for the aberrant CD34 isoforms in ZNF384-r (n = 32) B-ALL in our cohort, which is also characterized by high CD34 expression.According to the RNA-seq based pseudo-time analysis of our B-ALL cohort, IGH-DUX4 cases mostly tended to be common-B ALL, while ZNF384-r cases presented with more Pro-B ALL, which is also in accordance with the previous report based on immunophenotype analysis. To further discover the discrepancy between DUX4-r and ZNF384-r B-ALL subtypes, we compared DEGs and functional enrichment analysis between these two subtypes with B-other ALL as control, respectively (Figure 2). Activation of JAK-STAT signaling and downregulation of SOCS2, which acts as a negative controller of JAK-STAT signaling, were observed in both subtypes (Figure 2). The hematopoietic cell lineage gene set was enriched in ZNF384-r, consistent with its more immature phenotype. The co-expression gene network for IGH-DUX4 was constructed, and the hub gene APELA was emphasized among the closely related genes (Figure 3).DiscussionIn this study, we firstly identified two novel in-frame CD34 isoforms that share a common alternative first exon at its 5'UTR and are highly enriched in the IGH-DUX4 subtype B-ALL. It has been reported that intragenic focal ERG deletion is characteristically co-occurred with IGH-DUX4, and ERG targets CD34 through regulates its super-enhancer to affect the transcription program. It needs further investigation whether ERG deletion contributes to the aberrant transcription of CD34 in IGH-DUX4 subtype B-ALL. We also emphasized APELA as a hub gene in the transcription regulation network of IGH-DUX4. As it currently lacks effective targeted therapy for IGH-DUX4 subtype B-ALL, the potential therapeutic significance of the closely accompanied aberrant CD34 isoforms and APELA as a hub gene deserves further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The chromatin insulator CTCF regulates HPV18 transcript splicing and differentiation-dependent late gene expression.

The ubiquitous host protein, CCCTC-binding factor (CTCF), is an essential regulator of cellular transcription and functions to maintain epigenetic boundaries, stabilise chromatin loops and regulate splicing of alternative exons. We have previously demonstrated that CTCF binds to the E2 open reading frame (ORF) of human papillomavirus (HPV) 18 and functions to repress viral oncogene expression in undifferentiated keratinocytes by co-ordinating an epigenetically repressed chromatin loop within HPV episomes. Keratinocyte differentiation disrupts CTCF-dependent chromatin looping of HPV18 episomes promoting induction of enhanced viral oncogene expression. To further characterise CTCF function in HPV transcription control we utilised direct, long-read Nanopore RNA-sequencing which provides information on the structure and abundance of full-length transcripts. Nanopore analysis of primary human keratinocytes containing HPV18 episomes before and after synchronous differentiation allowed quantification of viral transcript species, including the identification of low abundance novel transcripts. Comparison of transcripts produced in wild type HPV18 genome-containing cells to those identified in CTCF-binding deficient genome-containing cells identifies CTCF as a key regulator of differentiation-dependent late promoter activation, required for efficient E1^E4 and L1 protein expression. Furthermore, our data show that CTCF binding at the E2 ORF promotes usage of the downstream weak splice donor (SD) sites SD3165 and SD3284, to the dominant E4 splice acceptor site at nucleotide 3434. These findings demonstrate that in the HPV life cycle both early and late virus transcription programmes are facilitated by recruitment of CTCF to the E2 ORF.

Dynamic Variations of 3'UTR Length Reprogram the mRNA Regulatory Landscape.

This paper concerns 3′-untranslated regions (3′UTRs) of mRNAs, which are non-coding regulatory platforms that control stability, fate and the correct spatiotemporal translation of mRNAs. Many mRNAs have polymorphic 3′UTR regions. Controlling 3′UTR length and sequence facilitates the regulation of the accessibility of functional effectors (RNA binding proteins, miRNAs or other ncRNAs) to 3′UTR functional boxes and motifs and the establishment of different regulatory landscapes for mRNA function. In this context, shortening of 3′UTRs would loosen miRNA or protein-based mechanisms of mRNA degradation, while 3′UTR lengthening would strengthen accessibility to these effectors. Alterations in the mechanisms regulating 3′UTR length would result in widespread deregulation of gene expression that could eventually lead to diseases likely linked to the loss (or acquisition) of specific miRNA binding sites. Here, we will review the mechanisms that control 3′UTR length dynamics and their alterations in human disorders. We will discuss, from a mechanistic point of view centered on the molecular machineries involved, the generation of 3′UTR variability by the use of alternative polyadenylation and cleavage sites, of mutually exclusive terminal alternative exons (exon skipping) as well as by the process of exonization of Alu cassettes to generate new 3′UTRs with differential functional features.

Mapping and modeling the genomic basis of differential RNA isoform expression at single-cell resolution with LR-Split-seq

The rise in throughput and quality of long-read sequencing should allow unambiguous identification of full-length transcript isoforms. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here we develop and characterize long-read Split-seq (LR-Split-seq), which uses combinatorial barcoding to sequence single cells with long reads. Applied to the C2C12 myogenic system, LR-split-seq associates isoforms to cell types with relative economy and design flexibility. We find widespread evidence of changing isoform expression during differentiation including alternative transcription start sites (TSS) and/or alternative internal exon usage. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells.

Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

CD44v6 Defines a New Population of Circulating Tumor Cells Not Expressing EpCAM.

Simple SummaryIn the present work, we describe (for the first time) the use of the transmembrane protein, CD44v6, to detect CTCs from blood samples of several patients with colorectal or breast cancer. We used CD44v6 antibodies to demonstrate that live CTCs can be specifically purified from CRC patient blood samples via magnetic bead- or FACS-based isolation techniques. Finally, we demonstrated that CD44v6-positive CTCs rarely expressed EpCam, which is currently the gold standard to enumerate CTCs, suggesting the need to use a combination of markers for a more comprehensive view of CTC heterogeneity.Circulating tumor cells (CTCs) are promising diagnostic and prognostic tools for clinical use. In several cancers, including colorectal and breast, the CTC load has been associated with a therapeutic response as well as progression-free and overall survival. However, counting and isolating CTCs remains sub-optimal because they are currently largely identified by epithelial markers such as EpCAM. New, complementary CTC surface markers are therefore urgently needed. We previously demonstrated that a splice variant of CD44, CD44 variable alternative exon 6 (CD44v6), is highly and specifically expressed by CTC cell lines derived from blood samples in colorectal cancer (CRC) patients. Two different approaches—immune detection coupled with magnetic beads and fluorescence-activated cell sorting—were optimized to purify CTCs from patient blood samples based on high expressions of CD44v6. We revealed the potential of the CD44v6 as a complementary marker to EpCAM to detect and purify CTCs in colorectal cancer blood samples. Furthermore, this marker is not restricted to colorectal cancer since CD44v6 is also expressed on CTCs from breast cancer patients. Overall, these results strongly suggest that CD44v6 could be useful to enumerate and purify CTCs from cancers of different origins, paving the way to more efficacious combined markers that encompass CTC heterogeneity.

Alternative splicing regulation of cell-cycle genes by SPF45/SR140/CHERP complex controls cell proliferation.

The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation, but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140, and CHERP, form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons flanked by suboptimal 3′ splice sites. These comprise alternative exons embedded in genes with important functions in cell-cycle progression, including the G2/M key regulator FOXM1 and the spindle regulator SPDL1. Knockdown of either of the three factors leads to G2/M arrest and to enhanced apoptosis in HeLa cells. Promoting the changes in FOXM1 or SPDL1 splicing induced by SPF45/SR140/CHERP knockdown partially recapitulates the effects on cell growth, arguing that the complex orchestrates a program of alternative splicing necessary for efficient cell proliferation.

DMD - BRAIN: EP.134 Regulation of the alternative splicing of Dp71, the major brain dystrophin transcript, by splicing factors

Duchenne muscular dystrophy (DMD) is a fatal childhood genetic disorder, caused by the absence of dystrophin throughout the body. Parallel to muscle degradation, loss of dystrophin in the brain can lead to cognitive impairments and intellectual disabilities. Such DMD brain co-morbidities are linked to the loss of dystrophin protein 71 (Dp71). Dp71 is the most predominant dystrophin protein in the brain, and alternative splicing at exons 71 and 78 produces several Dp71 isoforms with different subcellular locations and functions. Splicing factors regulating DMD alternative exons in skeletal muscle have recently been identified but it is not known what complement of splicing factors regulate DMD alternative splicing in the brain. Given the importance of Dp71 in the brain, we aim to assess the neuronal splicing factors regulating the alternative splicing of Dp71 exons 71 and 78 in the brain and whether this is disrupted in DMD. We have generated DMD minigenes to study the alternative splicing of exons 71 and 78. These are being used to assess the activity of neuronal splicing factors such as the CUG-BP and ETR-3-like factor (CELF) and muscleblind-like (MBNL) families on the alternative splicing of Dp71. Additionally, we are modelling the loss of Dp71 in neuronal and glial cell lines, and DMD patient-derived fibroblasts with distal DMD mutations and assessing the effect of Dp71 loss on the expression of CELF and MBNL splicing factors. Data from this work will be presented which provides a preliminary understanding of the significance of DMD alternative splicing within the brain. The development of brain-targeting treatments is complicated by the added complexity of dystrophin biology in the brain. Our work aims to inform such work through increasing our understanding of Dp71.

FGF-2 promotes angiogenesis through a SRSF1/SRSF3/SRPK1-dependent axis that controls VEGFR1 splicing in endothelial cells

BackgroundAngiogenesis is the process by which new blood vessels arise from pre-existing ones. Fibroblast growth factor-2 (FGF-2), a leading member of the FGF family of heparin-binding growth factors, contributes to normal as well as pathological angiogenesis. Pre-mRNA alternative splicing plays a key role in the regulation of cellular and tissular homeostasis and is highly controlled by splicing factors, including SRSFs. SRSFs belong to the SR protein family and are regulated by serine/threonine kinases such as SRPK1. Up to now, the role of SR proteins and their regulators in the biology of endothelial cells remains elusive, in particular upstream signals that control their expression.ResultsBy combining 2D endothelial cells cultures, 3D collagen sprouting assay, a model of angiogenesis in cellulose sponges in mice and a model of angiogenesis in zebrafish, we collectively show that FGF-2 promotes proliferation, survival, and sprouting of endothelial cells by activating a SRSF1/SRSF3/SRPK1-dependent axis. In vitro, we further demonstrate that this FGF-2-dependent signaling pathway controls VEGFR1 pre-mRNA splicing and leads to the generation of soluble VEGFR1 splice variants, in particular a sVEGFR1-ex12 which retains an alternative last exon, that contribute to FGF-2-mediated angiogenic functions. Finally, we show that sVEGFR1-ex12 mRNA level correlates with that of FGF-2/FGFR1 in squamous lung carcinoma patients and that sVEGFR1-ex12 is a poor prognosis marker in these patients.ConclusionsWe demonstrate that FGF-2 promotes angiogenesis by activating a SRSF1/SRSF3/SRPK1 network that regulates VEGFR1 alternative splicing in endothelial cells, a process that could also contribute to lung tumor progression.

Nanopore sequencing reveals endogenous NMD-targeted isoforms in human cells

BackgroundNonsense-mediated mRNA decay (NMD) is a eukaryotic, translation-dependent degradation pathway that targets mRNAs with premature termination codons and also regulates the expression of some mRNAs that encode full-length proteins. Although many genes express NMD-sensitive transcripts, identifying them based on short-read sequencing data remains a challenge.ResultsTo identify and analyze endogenous targets of NMD, we apply cDNA Nanopore sequencing and short-read sequencing to human cells with varying expression levels of NMD factors. Our approach detects full-length NMD substrates that are highly unstable and increase in levels or even only appear when NMD is inhibited. Among the many new NMD-targeted isoforms that our analysis identifies, most derive from alternative exon usage. The isoform-aware analysis reveals many genes with significant changes in splicing but no significant changes in overall expression levels upon NMD knockdown. NMD-sensitive mRNAs have more exons in the 3΄UTR and, for those mRNAs with a termination codon in the last exon, the length of the 3΄UTR per se does not correlate with NMD sensitivity. Analysis of splicing signals reveals isoforms where NMD has been co-opted in the regulation of gene expression, though the main function of NMD seems to be ridding the transcriptome of isoforms resulting from spurious splicing events.ConclusionsLong-read sequencing enables the identification of many novel NMD-sensitive mRNAs and reveals both known and unexpected features concerning their biogenesis and their biological role. Our data provide a highly valuable resource of human NMD transcript targets for future genomic and transcriptomic applications.

LABRAT reveals association of alternative polyadenylation with transcript localization, RNA binding protein expression, transcription speed, and cancer survival

BackgroundThe sequence content of the 3′ UTRs of many mRNA transcripts is regulated through alternative polyadenylation (APA). The study of this process using RNAseq data, though, has been historically challenging.ResultsTo combat this problem, we developed LABRAT, an APA isoform quantification method. LABRAT takes advantage of newly developed transcriptome quantification techniques to accurately determine relative APA site usage and how it varies across conditions. Using LABRAT, we found consistent relationships between gene-distal APA and subcellular RNA localization in multiple cell types. We also observed connections between transcription speed and APA site choice as well as tumor-specific transcriptome-wide shifts in APA isoform abundance in hundreds of patient-derived tumor samples that were associated with patient prognosis. We investigated the effects of APA on transcript expression and found a weak overall relationship, although many individual genes showed strong correlations between relative APA isoform abundance and overall gene expression. We interrogated the roles of 191 RNA-binding proteins in the regulation of APA isoforms, finding that dozens promote broad, directional shifts in relative APA isoform abundance both in vitro and in patient-derived samples. Finally, we find that APA site shifts in the two classes of APA, tandem UTRs and alternative last exons, are strongly correlated across many contexts, suggesting that they are coregulated.ConclusionsWe conclude that LABRAT has the ability to accurately quantify APA isoform ratios from RNAseq data across a variety of sample types. Further, LABRAT is able to derive biologically meaningful insights that connect APA isoform regulation to cellular and molecular phenotypes.

A CRISPR RNA-Binding Protein Screen Reveals Novel Regulators of RUNX1 Alternative Polyadenylation

Alternative polyadenylation (APA) is a crucial post-transcriptional process that regulates the 3' end formation of over 75% of all human genes. Despite its prevalence and importance during development and in disease, there are few studies that focus on APA in the hematology field. Interestingly, APA affects the coding sequence of RUNX1, a pivotal transcription factor for proper hematopoiesis. Usage of the most proximal RUNX1 polyadenylation sequence (PAS) in upstream alternative terminal exon 7a results in the C-terminally truncated RUNX1a isoform. RUNX1a uniquely promotes self-renewal and expansion of hematopoietic stem cells (HSCs). In the endogenous context, usage of this proximal PAS is tightly restricted, resulting in polyadenylation at one of three distal PAS in exon 8 and producing the RUNX1b/c isoforms. RUNX1b/c are much more abundant and promote HSC differentiation and lineage commitment. Importantly, the ratio of RUNX1a to RUNX1b/c is developmentally-regulated. Therefore, it is highly significant to uncover the APA mechanism that controls the RUNX1 isoform ratio and consequently the balance of HSC self-renewal and differentiation. We hypothesize that there are enhancers and suppressors of the proximal RUNX1 PAS, tightly controlling the relative amount of RUNX1a produced during hematopoiesis.To identify RUNX1 APA regulators, we first developed a strategy to monitor RUNX1 proximal PAS usage in vitro. We cloned alternative terminal exon 7a, which contains the proximal PAS, along with 500 bp of up and downstream flanking intron between the two exons of a split GFP reporter. When we introduce this fluorescent minigene reporter into blood cells, two transcripts can be produced: (1) the first GFP exon splices and polyadenylates RUNX1 exon 7a, producing no GFP (RUNX1a formation) or (2) the two GFP exons are spliced together, skipping the proximal PAS in exon 7a, producing GFP (RUNX1b/c formation). Since RUNX1b/c are much more highly expressed than RUNX1a throughout hematopoiesis, we observed a robust GFP signal when this reporter was transfected into MDS-L, K562, and U937 cell lines. RT-PCR analysis confirmed that both transcripts are produced, albeit at dramatically different levels. We can thus use GFP fluorescence as a read-out for RUNX1 proximal PAS usage.Next, we generated a stable, clonal MDS-L cell line expressing this RUNX1 exon 7a split GFP reporter. We used this reporter line to perform a CRISPR screen using an sgRNA library that is enriched for targeting RNA-binding proteins (RBPs). We infected cells with the library, cultured in puromycin to force sgRNA and Cas9 integration, waited three weeks to ensure gene knockout, and then used fluorescence-activated cell sorting (FACS) to isolate GFP low and high cell populations. GFP low cells contained sgRNAs targeting RUNX1a suppressors since these cells exhibited enhanced proximal PAS usage. Conversely, GFP high cells contained sgRNAs targeting RUNX1a enhancers since proximal PAS usage was further diminished. We identified 44 putative RUNX1a suppressors and 41 putative enhancers (p < 0.05).We then sought to validate the genes revealed in our CRISPR screen by using individual sgRNAs targeting a subset of the candidates. One confirmed suppressor is HNRNPA1, an RBP which is upregulated and leukemogenic in BCR/ABL chronic myelogenous leukemia (CML). In HNRNPA1 knockout cells, there was a 5-fold increase in relative RUNX1a and a corresponding decrease in RUNX1b/c levels by RT-qPCR. This effect was dose-dependent as heterozygous HNRNPA1 cells exhibited an intermediate 2.5-fold increase in RUNX1a mRNA. We verified this data by performing shRNA knockdown of HNRNPA1 in MDS-L and K562 cells, again observing a significant increase in relative RUNX1a levels. Interestingly, HNRNPA1 is highly expressed throughout the course of hematopoiesis, indicating that a cooperating protein helps facilitate this suppressive effect. Indeed, numerous HNRNPA1-interacting proteins were also identified in our screen and are the subject of current studies.In summary, we devised a fluorescent minigene reporter to monitor RUNX1 APA in vitro. Using this reporter, we performed a CRISPR RBP screen and identified HNRNPA1 as the founding member of a complex that suppresses RUNX1a production during hematopoiesis. HNRNPA1 and additional regulators represent novel proteins that have the potential to regulate HSC behavior by modifying the RUNX1 isoform ratio. DisclosuresNo relevant conflicts of interest to declare.

Unique post-translational modifications diversify the sodium channels in peach aphid (Myzus persicae [Sulzer]).

Myzus persicae (Sulzer), a worldwide pest, has caused remarkable damage to agriculture. Among the various control methods, chemical control (especially pyrethroids) is most commonly used. The targets of pyrethroids are voltage-gated sodium channels (Nav s). Unlike those of other insects, all Nav s of aphids (including two genes), such as Myzus persicae, are unique. In this study, three interlock patterns, I(918)-F(1014), L(918)-L(1014), and T(918)-F(1014), were found at sites 918 and 1014 in the sensitive Myzus persicae strain. Similar to that of other aphids, the Nav of Myzus persicae (MpNav ) consisted of two parts, that is MpNav -I and MpNav -II, which were embedded with an atypical 'DENS' ion selectivity filter and a conventional 'MFM' inactivation gate, respectively. MpNav had 11 alternative exons, including two mutually exclusive exons (k and l) and three exons (w, x, and t), which were located in domains I and III, respectively. In addition, various RNA editing events, A503T and V588A, appearing between the connection of domains I and II and the S3 of domain IV, respectively, had been described. Overall, MpNav was characterized by unique post-translational regulation mode, 918 and 1014 interlocks, and unusually alternative exons. Our research provides a new perspective on the evolution and variation of insect Nav s. © 2021 Society of Chemical Industry.

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants.

Simple SummaryIn the BRIDGES project, the breast/ovarian cancer gene RAD51D has been sequenced in >113,000 women. In the present study, we focused on the impact that 11 pre-selected RAD51D variants at the intron/exon boundaries had on the splicing process (intron removal). For this purpose, we developed a splicing reporter minigene, containing RAD51D-exons 2–9 wherein any variant could be introduced and functionally assayed for splicing alterations. All variants impaired splicing, 10 of which caused complete splicing aberrations. Moreover, we developed a minigene-based strategy to search for non-canonical, spliceogenic variants that disrupted splicing enhancers/silencers in the non-constitutive exon 3. Twenty-six BRIDGES and 16 artificial exon 3 variants were also tested. Thirty variants impaired splicing by producing variable amounts of the FL transcript. In total, up to 9 variants were classified as Likely Pathogenic, and therefore were clinically actionable. Carriers may benefit from tailored prevention protocols and therapies.RAD51D loss-of-function variants increase lifetime risk of breast and ovarian cancer. Splicing disruption is a frequent pathogenic mechanism associated with variants in susceptibility genes. Herein, we have assessed the splicing and clinical impact of splice-site and exonic splicing enhancer (ESE) variants identified through the study of ~113,000 women of the BRIDGES cohort. A RAD51D minigene with exons 2–9 was constructed in splicing vector pSAD. Eleven BRIDGES splice-site variants (selected by MaxEntScan) were introduced into the minigene by site-directed mutagenesis and tested in MCF-7 cells. The 11 variants disrupted splicing, collectively generating 25 different aberrant transcripts. All variants but one produced negligible levels (<3.4%) of the full-length (FL) transcript. In addition, ESE elements of the alternative exon 3 were mapped by testing four overlapping exonic microdeletions (≥30-bp), revealing an ESE-rich interval (c.202_235del) with critical sequences for exon 3 recognition that might have been affected by germline variants. Next, 26 BRIDGES variants and 16 artificial exon 3 single-nucleotide substitutions were also assayed. Thirty variants impaired splicing with variable amounts (0–65.1%) of the FL transcript, although only c.202G>A demonstrated a complete aberrant splicing pattern without the FL transcript. On the other hand, c.214T>C increased efficiency of exon 3 recognition, so only the FL transcript was detected (100%). In conclusion, 41 RAD51D spliceogenic variants (28 of which were from the BRIDGES cohort) were identified by minigene assays. We show that minigene-based mapping of ESEs is a powerful approach for identifying ESE hotspots and ESE-disrupting variants. Finally, we have classified nine variants as likely pathogenic according to ACMG/AMP-based guidelines, highlighting the complex relationship between splicing alterations and variant interpretation.

ENTPD5: identification of splicing variants and their impact on cancer survival

NTPDase5 is a nucleotidase of the endoplasmic reticulum that plays an important role in proteostasis as a regulator of protein N-glycosylation. This enzyme was first identified in hamster as a proto-oncogene activated upon a single nucleotide deletion that causes a frameshift leading to a truncated protein. Truncated NTPDase5 proteins were detected in human samples, but an oncogene was never identified. Searching for transcript variants in the GenBank database and using TCGA data, we discovered that splice variants could originate truncated human NTPDase5 proteins. We identified three main splicing events in the ENTPD5 gene: alternative acceptors, exon skipping, and alternative terminators. The analysis of impact of splicing events in cancers showed that skipping of exon 11-the event that leads to truncated proteins similar in size to the hamster oncogene-does not affect the hazard ratio of most tumors and was, in fact, a protective factor in the only two cancer studies where it was significant. We also identified four main patterns of impact of ENTPD5 in cancer and a potential variant-specific regulation by miR-215. Our findings shed light on a two-decade uncertainty about the origin of truncated NTPDase5 and contribute to the characterization of its impacts in cancer.

Aerobic Exercise Induces Alternative Splicing of Neurexins in Frontal Cortex.

Aerobic exercise (AE) is known to produce beneficial effects on brain health by improving plasticity, connectivity, and cognitive functions, but the underlying molecular mechanisms are still limited. Neurexins (Nrxns) are a family of presynaptic cell adhesion molecules that are important in synapsis formation and maturation. In vertebrates, three-neurexin genes (NRXN1, NRXN2, and NRXN3) have been identified, each encoding for α and β neurexins, from two independent promoters. Moreover, each Nrxns gene (1–3) has several alternative exons and produces many splice variants that bind to a large variety of postsynaptic ligands, playing a role in trans-synaptic specification, strength, and plasticity. In this study, we investigated the impact of a continuous progressive (CP) AE program on alternative splicing (AS) of Nrxns on two brain regions: frontal cortex (FC) and hippocampus. We showed that exercise promoted Nrxns1–3 AS at splice site 4 (SS4) both in α and β isoforms, inducing a switch from exon-excluded isoforms (SS4−) to exon-included isoforms (SS4+) in FC but not in hippocampus. Additionally, we showed that the same AE program enhanced the expression level of other genes correlated with synaptic function and plasticity only in FC. Altogether, our findings demonstrated the positive effect of CP AE on FC in inducing molecular changes underlying synaptic plasticity and suggested that FC is possibly a more sensitive structure than hippocampus to show molecular changes.