A cell-to-patient machine learning transfer approach uncovers novel basal-like breast cancer prognostic markers amongst alternative splice variants
Jean-Philippe VilleminClaudio LorenziMarie-Sarah CabrillacAndrew J. OldfieldWilliam RitchieReini F. Luco
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Abstract:
Breast cancer is amongst the 10 first causes of death in women worldwide. Around 20% of patients are misdiagnosed leading to early metastasis, resistance to treatment and relapse. Many clinical and gene expression profiles have been successfully used to classify breast tumours into 5 major types with different prognosis and sensitivity to specific treatments. Unfortunately, these profiles have failed to subclassify breast tumours into more subtypes to improve diagnostics and survival rate. Alternative splicing is emerging as a new source of highly specific biomarkers to classify tumours in different grades. Taking advantage of extensive public transcriptomics datasets in breast cancer cell lines (CCLE) and breast cancer tumours (TCGA), we have addressed the capacity of alternative splice variants to subclassify highly aggressive breast cancers.Keywords:
Basal (medicine)
Triple-negative breast cancer
ABSTRACT Understanding regulation of MAPT splicing is important to the etiology of many nerurodegenerative diseases, including Alzheimer disease (AD) and progressive supranuclear palsy (PSP), in which different tau isoforms accumulate in pathologic inclusions. MAPT , the gene encoding the tau protein, undergoes complex alternative pre-mRNA splicing to generate six isoforms. Tauopathies can be categorized by the presence of tau aggregates containing either 3 (3R) or 4 (4R) microtubule binding domain repeats (determined by inclusion/exclusion of exon 10), but the role of the N terminal domain of the protein, determined by inclusion/exclusion of exons 2 and 3 has been less well studied. Using an unbiased correlational screen in human brain tissue, we observed coordination of MAPT exons 2 and 10 splicing. Expression of exon 2 splicing regulators and subsequently exon 2 inclusion are differentially disrupted in PSP and AD brain, resulting in the accumulation of 1N4R isoforms in PSP and 0N isoforms in AD temporal cortex. Furthermore, we identified different N-terminal isoforms of tau present in neurofibrillary tangles, dystrophic neurites and tufted astrocytes, indicating a role for differential N-terminal splicing in the development of disparate tau neuropathologies. We conclude that N-terminal splicing and combinatorial regulation with exon 10 inclusion/exclusion is likely to be important to our understanding of tauopathies.
Tauopathy
Neurite
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Voltage-gated sodium channels are the primary molecules responsible for the rising phase of action potentials in electrically excitable cells. There are 10 distinct sodium channel isoforms Nav 1.1-1.8 (SCNIA-SCN5A and SCN8A-SCNIIA) and the majority of these undergo tissue and developmentally regulated alternative splicing. Two such examples are those of the SCN8A (Nav 1.6) and SCN9A (Nav 1.7) genes. SCN8A gene contains two mutually exclusive exons, 18N and 18A. Transcripts with exon 18N have a conserved inframe stop codon that predicts the synthesis of a truncated, non functional sodium channel. This protein is expressed in fetal brain and non-neuronal tissues. Once the exon 18A is included, the resulted protein will be a functional channel, that is expressed in adult neurons ofCNS and PNS. The SCN9A exon 5N is preferentially expressed in the PNS and CNS of adult tissues and significant usage of exon 5A was found only in DRG. These two isoforms differ in one amino acid in the S3 domain I (exons 5A and 5N). This change of one amino acid induced a small shift of activation to more hyperpolarized potentials forexon SA compared with exon SN. Analysis of SeNSA pre-mRNA splicing supports a model in which exon 18A exclusion in non-neuronal tissue is regulated primarily by the presence in the cell types of several hnRNPs proteins that function through an exonic splicing silencer (ESS) found in this exon together with the absence of neuron specific Fox-I protein. In neuronal cells the absence of these hnRNPs together with the presence of neuron specific Fox-l cause the exon to be included. The SeNSA exon 18N is included innon neuronal cells due to the SR proteins that function through an exonic splicing enhancer(ESE) found in this exon. In neuronal cells the lower levels of these SR proteins cause the exon 18N to be skipped. This type of control of mutually exclusive splicing through the proteome make-up of a cell type would appear to be influential in the temporal and tissue specific splicing of SeN8A, another member of the voltage gated sodium channels and may indeed represent a more general mechanism.
Exon trapping
Stop codon
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Alternative splicing of tau exon 10 generates tau isoforms with 3 or 4 microtubule-binding repeats.In normal adult human brain,approximately equal levels of 3R-tau and 4R-tau are expressed,which is required for maintaining normal brain functions.9G8,one member of SR protein family,is involved in the splicing of many genes.The function of 9G8 is highly regulated by the phosphorylation.It was reported that GSK-3β regulates the alternative splicing of tau exon 10.Mini-tau gene were used to study the regulation of 9G8 on tau exon 10 splicing and the effect of GSK-3β on 9G8-mediated tau exon 10 splicing.It was found that 9G8 inhibited the inclusion of tau exon 10.GSK-3β phosphorylated 9G8 in vitro and interacted with 9G8.Overexpression of GSK-3β inhibited 9G8-mediated tau exon 10 inclusion.
Minigene
Exonic splicing enhancer
Tau protein
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IL-15 promotes the growth of T cells and shares properties of IL-2. IL-2 is produced exclusively by T cells, while IL-15 message is expressed by a variety of tissues. However, it has been difficult to demonstrate IL-15 in the supernatants of many cells that express message for this cytokine. This suggests that IL-15 production is regulated by post-transcriptional controls. In this study, we cloned three types of murine IL-15 cDNA isoforms generated by alternative splicing and compared the translational efficiency among these isoforms. The translational efficiency of isoforms with alternative exon 5 containing another 3' splice site was significantly higher than that of IL-15 cDNA with originally described exon 5, which is generated by internal splicing of alternative exon 5. The translation product of the isoform containing alternative exon 5 has a shorter open reading frame due to stop codons in additional sequence, followed by a new AUG codon, and displays a shorter leader sequence. The shorter isoform of the IL-15 was detected in peritoneal macrophages stimulated with IFN-gamma and LPS, which expressed an abundant level of alternative exon 5. These results suggest that normal IL-15 production in stimulated macrophages is regulated by splicing of alternative exon 5.
Translational efficiency
Exon trapping
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Abstract The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.
Exonic splicing enhancer
Exon trapping
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CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons. The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure. Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.
RNA recognition motif
Exon skipping
Exonic splicing enhancer
Splicing factor
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NF-YA, the regulatory subunit of the trimeric CCAAT-binding transcription factor NF-Y, is present in vertebrates in two major alternative spliced isoforms: NF-YAl and NF-YAs, differing for the presence of exon-3. NF-YAx, a third isoform without exon-3/-5, was reported only in human neuronal cells and tumors. These events affect the Trans-Activation Domain. We provide here evidence for the expression of NF-YAx and for the existence of a new isoform, NF-YAg, skipping only exon-5. These isoforms are abundant in Aves, but not in reptiles, and are the prevalent transcripts in the initial phases of embryo development in chicken. Finally, we analyzed NF-YAg and NF-YAx amino acid sequence using AlphaFold: absence of exon-5 denotes a global reduction of β-stranded elements, while removal of the disordered exon-3 sequence has limited effects on TAD architecture. These data identify an expanded program of NF-YA isoforms within the TAD in Aves, implying a role during early development.
Transcription
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Alternative splicing events that are conserved in orthologous genes in different species are commonly viewed as reliable evidence of authentic, functionally significant alternative splicing events. Several recent bioinformatic analyses have shown that conserved alternative exons possess several features that distinguish them from alternative exons that are species-specific. One of the most striking differences between conserved and species-specific alternative exons is the high percentage of exons that preserve the reading frame (exons whose length is an exact multiple of 3, termed symmetrical exons) among the conserved alternative exons. Here, we examined conserved alternative exons and found several features that differentiate between symmetrical and non-symmetrical alternative exons. We show that symmetrical alternative exons have a strong tendency not to disrupt protein domain structures, whereas the tendency of non-symmetrical alternative exons to overlap with different fractions of protein domains is similar to that of constitutive exons. Additionally, skipping isoforms of non-symmetrical alternative exons are strongly underrepresented, compared with their including isoforms, suggesting that skipping of a large fraction of non-symmetrical alternative exons produces transcripts that are degraded by the nonsense-mediated mRNA decay mechanism. Non-symmetrical alternative exons also show a tendency to reside in the 5′ half of the CDS. These findings suggest that alternative splicing of symmetrical and non-symmetrical exons is governed by different selective pressures and serves different purposes.
Exon shuffling
Exon trapping
Exon skipping
Trans-splicing
Exonic splicing enhancer
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An alternatively spliced TREM2 isoform lacking the ligand binding domain is expressed in human brain
Abstract Background Genetic variants in TREM2 are strongly associated with Alzheimer’s Disease (AD) risk but alternative splicing in TREM2 transcripts has not been comprehensively described. Objective Recognizing that alternative splice variants can result in reduced gene expression and/or altered function, we sought to fully characterize splice variation in TREM2 . Methods Human blood and anterior cingulate autopsy tissue from 61 donors were used for end-point and quantitative PCR and Western blotting to identify and quantify novel TREM2 isoforms. Results In addition to previously described transcripts lacking exon 3 or exon 4, or retaining part of intron 3, we identified novel isoforms lacking exon 2, along with isoforms lacking multiple exons. Isoforms lacking exon 2 were predominant at approximately 10% of TREM2 mRNA in the brain. Expression of TREM2 and frequency of exon 2 skipping did not differ between AD samples and non-AD controls (p = 0.1268 and p = 0.4909, respectively). Further, these novel splice isoforms were also observed across multiple tissues with similar frequency (range 5.3 – 13.0%). We found that the exon 2 skipped isoform D2-TREM2 is translated to protein and localizes similarly to full-length TREM2 protein, that both proteins are primarily retained in the Golgi complex, and that D2-TREM2 is expressed in AD and non-AD brain. Conclusion Since the TREM2 ligand binding domain is encoded by exon 2, and skipping this exon retains reading frame while conserving localization, we hypothesize that D2-TREM2 acts as an inhibitor of TREM2 and targeting TREM2 splicing may be a novel therapeutic pathway for AD.
TREM2
Exon skipping
Protein isoform
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