Alternative Splicing Of Several Genes Research Articles

Lung Transcriptomics Links Emphysema to Barrier Dysfunction and Macrophage Subpopulations.

While many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient non-imaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort. Our goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung samples. Using RNA-sequencing data from 446 samples in the Lung Tissue Research Consortium (LTRC) and 3,606 blood samples from the COPDGene study, we examined the transcriptomic features of chest computed tomography-quantified emphysema. We also leveraged publicly available lung single-cell RNA-sequencing data to identify cell types showing COPD-associated differential expression of the emphysema pathways found in the bulk analyses. In the bulk lung RNA-seq analysis, 1,087 differentially expressed genes and 34 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of several genes and increased activity in pluripotency and cell barrier function pathways. Lung tissue and blood samples shared differentially expressed genes and biological pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and distinct pathway activities were observed among various macrophage subpopulations. This study identified emphysema-related changes in gene expression and alternative splicing, cell-type specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung.

The C2H2 family protein ZAT17 engages in the cadmium stress response by interacting with PRL1 in Arabidopsis

Cadmium (Cd) is a heavy metal and a toxic substance. Soil Cd pollution has emerged as a significant environmental issue that jeopardizes both the safety of agricultural products and human health. PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) has been identified as a crucial factor in Cd stress and a series of defence mechanisms. However, the mechanism through which PRL1 mediates its downstream signalling has remained poorly understood. Here, we discovered a prl1–2 suppressor (sup8) for prl1–2 that complemented the defective development phenotype of prl1–2 under Cd stress. Gene cloning revealed a mutation in the C2H2 transcription factor ZAT17 as the basis for the sup8 phenotype. Genetic and biochemical studies indicated that ZAT17 acts as a negative regulator of Cd tolerance. Transcriptome analysis revealed that ZAT17 influences the alternative splicing (AS) process of multiple Cd-responsive genes by interacting with members of the MAC splicing complex, including PRL1 and CDC5. In conclusion, the identification of the novel gene ZAT17 enriches the understanding of the Cd stress response pathway and provides a valuable candidate locus for breeding Cd-resistant plant varieties.

TGF-\u03b2-mediated epithelial\u2013mesenchymal transition and tumor-promoting effects in CMT64 cells are reflected in the transcriptomic signature of human lung adenocarcinoma

Epithelial–mesenchymal transition (EMT) is a cellular process during which epithelial cells acquire mesenchymal phenotypes. Cancer cells undergo EMT to acquire malignant features and TGF-β is a key regulator of EMT. Here, we demonstrate for the first time that TGF-β could elicit EMT in a mouse lung adenocarcinoma cell line. TGF-β signaling activation led to cell morphological changes corresponding to EMT and enhanced the expression of mesenchymal markers and EMT-associated transcription factors in CMT64 lung cancer cells. RNA-sequencing analyses revealed that TGF-β increases expression of Tead transcription factors and an array of Tead2 target genes. TGF-β stimulation also resulted in alternative splicing of several genes including Cd44, tight junction protein 1 (Tjp1), and Cortactin (Cttn). In parallel with EMT, TGF-β enhanced cell growth of CMT64 cells and promoted tumor formation in a syngeneic transplantation model. Of clinical importance, the expression of TGF-β-induced genes identified in CMT64 cells correlated with EMT gene signatures in human lung adenocarcinoma tissue samples. Furthermore, TGF-β-induced gene enrichment was related to poor prognosis, underscoring the tumor-promoting role of TGF-β signaling in lung adenocarcinoma. Our cellular and syngeneic transplantation model would provide a simple and useful experimental tool to study the significance of TGF-β signaling and EMT.

Transcriptome Analysis in a Primary Human Muscle Cell Differentiation Model for Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions leading to a complex pathology with a multisystemic phenotype that primarily affects the muscles and brain. Despite a multitude of information, especially on the alternative splicing of several genes involved in the pathology, information about additional factors contributing to the disease development is still lacking. We performed RNAseq and gene expression analyses on proliferating primary human myoblasts and differentiated myotubes. GO-term analysis indicates that in myoblasts and myotubes, different molecular pathologies are involved in the development of the muscular phenotype. Gene set enrichment for splicing reveals the likelihood of whole, differentiation stage specific, splicing complexes that are misregulated in DM1. These data add complexity to the alternative splicing phenotype and we predict that it will be of high importance for therapeutic interventions to target not only mature muscle, but also satellite cells.

Transcriptome profiling reveals the high incidence of hnRNPA1 exon 8 inclusion in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a malignancy that evolves through a multi-step process. Alternative splicing of several genes has been linked to the progression of the disease, but involvement of alternations in splicing profiles has not been reported. RNA-seq of peripheral blood mononuclear cell (PBMC) samples characterized the differentially expressed and spliced transcripts in five CML chronic phase (CP) and five blast phase (BP) patients, and five healthy controls. Global splicing alteration analysis detected 6474 altered splicing events altered between CML and healthy samples, including many of the previously reported splicing variants and showing a more profound altered splicing deregulation in BP samples. Functional clustering of differentially spliced genes in CP revealed a preferred enrichment relating to cell signaling, while the spliceosome pathway was most overrepresented in BP samples. One differentially spliced spliceosome gene hnRNPA1 showed two splice isoforms; the longer isoform contained exon 8 was preferentially expressed in the BP patients, and the short one excluding exon 8 was specific to healthy controls. Our findings suggested that alternative splicing deregulation played a central role during the progression of CML from CP to BP, and the longer isoform of hnRNPA1 might represent a diagnostic marker and therapeutic target for CML.

TDP-43 and NOVA-1 RNA-binding proteins as competitive splicing regulators of the schizophrenia-associated TNIK gene

The RNA-binding protein TDP-43, associated to amyotrophic lateral sclerosis and frontotemporal dementia, regulates the alternative splicing of several genes, including the skipping of TNIK exon 15. TNIK, a genetic risk factor for schizophrenia and causative for intellectual disability, encodes for a Ser/Thr kinase regulating negatively F-actin dynamics.Here we show that in the human adult nervous system TNIK exon 15 is mostly included compared to the other tissues and that, during neuronal differentiation of human induced pluripotent stem cells and of human neuroblastoma cells, TNIK exon 15 inclusion increases independently of TDP-43 protein content. By studying the possible molecular interplay of TDP-43 with brain-specific splicing factors, we found that the neuronal NOVA-1 protein competitively inhibits both TDP-43 and hnRNPA2/B1 skipping activity on TNIK by means of a RNA-dependent interaction and that this competitive mechanism is common to other TDP-43 RNA targets. We also show that the TNIK protein isoforms including/excluding exon 15 differently regulate cell spreading in non-neuronal cells and neuritogenesis in primary cortical neurons.Our data suggest a complex regulation between the ubiquitous TDP-43 and the neuron-specific NOVA-1 splicing factors in the brain that may help better understand the pathobiology of both neurodegenerative diseases and schizophrenia.

Alternative pre-mRNA splicing switch controls hESC pluripotency and differentiation

Alternative splicing (AS) of pre-mRNAs is a ubiquitous process in mammals that is tightly regulated in a cell type- and cell state-dependent manner. However, the details of how splicing is regulated to impact specific cell fate decisions remains incompletely understood. A study by Yamazaki and colleagues (pp. 1161-1174) in this issue of Genes & Development provides exciting new insight into the role and regulation of splicing in the maintenance of pluripotency of human embryonic stem cells (hESCs). In brief, they show that AS of several genes is robustly regulated upon differentiation of hESCs. One of these genes, T-cell factor 3 (TCF3), is regulated at least in part through the activity of heterogeneous nuclear ribonucleoproteins H1 and F (hnRNP H/F) to control the mutually exclusive expression of the encoded E12 and E47 transcription regulators. The investigators demonstrate that reduced expression of hnRNP H/F favors expression of E47, which in turn decreases E-cadherin expression to promote hESC differentiation. In contrast, high levels of hnRNP H/F induce expression of E12 to maintain pluripotency. Thus, this work provides at least one new link between AS and control of human stem cell fate and suggests a broader role of splicing in pluripotency.

Intragenic DNA methylation and BORIS-mediated cancer-specific splicing contribute to the Warburg effect

Aberrant alternative splicing and epigenetic changes are both associated with various cancers, but epigenetic regulation of alternative splicing in cancer is largely unknown. Here we report that the intragenic DNA methylation-mediated binding of Brother of Regulator of Imprinted Sites (BORIS) at the alternative exon of Pyruvate Kinase (PKM) is associated with cancer-specific splicing that promotes the Warburg effect and breast cancer progression. Interestingly, the inhibition of DNA methylation, BORIS depletion, or CRISPR/Cas9-mediated deletion of the BORIS binding site leads to a splicing switch from cancer-specific PKM2 to normal PKM1 isoform. This results in the reversal of the Warburg effect and the inhibition of breast cancer cell growth, which may serve as a useful approach to inhibit the growth of breast cancer cells. Importantly, our results show that in addition to PKM splicing, BORIS also regulates the alternative splicing of several genes in a DNA methylation-dependent manner. Our findings highlight the role of intragenic DNA methylation and DNA binding protein BORIS in cancer-specific splicing and its role in tumorigenesis.

Comparable roles of CD44v8-10 and CD44s in the development of bone metastases in a mouse model.

Cluster of differentiation (CD)44 has been implicated in cancer metastasis to bone. Clinical and experimental studies have suggested that the standard isoform of CD44 (CD44s) and the variant isoform of CD44 (CD44v) enhance metastasis. The present study examined the differential roles of CD44s and CD44v, particularly CD44v8-10, in the development of bone metastases. For this purpose, MDA-MB-231 human breast cancer cells and A549 human lung cancer cells were stably transduced with epithelial splicing regulatory protein 1 (ESRP1), which regulates the alternative splicing of several genes, including CD44. The introduction of ESRP1 induced a splicing switch from CD44s to CD44v, particularly to CD44v8-10, while the total amount of CD44 was rarely affected. However, ESRP1 did not significantly affect cell proliferation, migration, invasion or tumor sphere formation in vitro. Furthermore, ESRP1 did not cause significant differences in the development of bone metastases in a mouse model. As an alternative approach, cancer cells transduced with the CD44v8-10 gene were also established. The overexpression of CD44v8-10 in MCF-7 human breast cancer cells, which rarely express any isoform of CD44, promoted cell migration and sphere formation, whereas the overexpression of CD44v8-10 in MDA-MB-231 cells, which endogenously express high levels of CD44s, did not exert these effects. The results of the present study collectively suggest that the ability of CD44v8-10 to promote tumor aggressiveness and bone metastases is similar to that of CD44s. CD44v8-10 and CD44s may represent potential therapeutic targets for the treatment of bone metastases.

Progression of muscle histopathology but not of spliceopathy in myotonic dystrophy type 2

Myotonic dystrophy type 2 (DM2) is an autosomal dominant progressive disease involving skeletal and cardiac muscle and brain. It is caused by a tetranucleotide repeat within the first intron of the CNBP gene that leads to an alteration of the alternative splicing of several genes. To understand the molecular mechanisms that play a role in DM2 progression, the evolution of skeletal muscle histopathology and biomolecular findings in successive biopsies have been studied. Biceps brachii biopsies from 5 DM2 patients who underwent two successive biopsies at different years of age have been used. Muscle histopathology has been assessed on sections immunostained with fast or slow myosin. FISH in combination with MBNL1-immunofluorescence has been performed to evaluate ribonuclear inclusion and MBNL1 foci dimensions in myonuclei. Gene and protein expression and alteration of alternative splicing of several genes have been evaluated over time. All DM2 patients examined show a worsening of muscle histopathology and an increase of foci dimensions over time. The progressive worsening of myotonia in DM2 patients may be due to the decrease of CLCN1 mRNA observed in all patients examined. However, a worsening of alternative splicing alterations has not been evidenced over time. The data obtained in this study confirm that DM2 is a slow progression disease since histological and biomolecular alterations observed in skeletal muscle are minimal even after 10-year interval. The data indicate that muscle morphological alterations evolve more rapidly over time than the molecular changes thus indicating that muscle biopsy is a more sensitive tool than biomolecular markers to assess disease progression at muscle level.

Muscleblind-like1 undergoes ectopic relocation in the nuclei of skeletal muscles in myotonic dystrophy and sarcopenia

Muscleblind-like 1 (MBNL1) is an alternative splicing factor involved in postnatal development of skeletal muscles and heart in humans and mice, and its deregulation is known to be pivotal in the onset and development of myotonic dystrophy (DM). In fact, in DM patients this protein is ectopically sequestered into intranuclear foci, thus compromising the regulation of the alternative splicing of several genes. However, despite the numerous biochemical and molecular studies, scarce attention has been paid to the intranuclear location of MBNL1 outside the foci, although previous data demonstrated that in DM patients various splicing and cleavage factors undergo an abnormal intranuclear distribution suggestive of impaired RNA processing. Interestingly, these nuclear alterations strongly remind those observed in sarcopenia i.e., the loss of muscle mass and function which physiologically occurs during ageing. On this basis, in the present investigation the ultrastructural localization of MBNL1 was analyzed in the myonuclei of skeletal muscles from healthy and DM patients as well as from adult and old (sarcopenic) mice, in the attempt to elucidate possible changes in its distribution and amount. Our data demonstrate that in both dystrophic and sarcopenic muscles MBNL1 undergoes intranuclear relocation, accumulating in its usual functional sites but also ectopically moving to domains which are usually devoid of this protein in healthy adults. This accumulation/delocalization could contribute to hamper the functionality of the whole splicing machinery, leading to a lower nuclear metabolic activity and, consequently, to a less efficient protein synthesis. Moreover, the similar nuclear alterations found in DM and sarcopenia may account for the similar muscle tissue features (myofibre atrophy, fibre size variability and centrally located nuclei), and, in general, for the aging-reminiscent phenotype observed in DM patients.

An EMT–Driven Alternative Splicing Program Occurs in Human Breast Cancer and Modulates Cellular Phenotype

Epithelial-mesenchymal transition (EMT), a mechanism important for embryonic development, plays a critical role during malignant transformation. While much is known about transcriptional regulation of EMT, alternative splicing of several genes has also been correlated with EMT progression, but the extent of splicing changes and their contributions to the morphological conversion accompanying EMT have not been investigated comprehensively. Using an established cell culture model and RNA–Seq analyses, we determined an alternative splicing signature for EMT. Genes encoding key drivers of EMT–dependent changes in cell phenotype, such as actin cytoskeleton remodeling, regulation of cell–cell junction formation, and regulation of cell migration, were enriched among EMT–associated alternatively splicing events. Our analysis suggested that most EMT–associated alternative splicing events are regulated by one or more members of the RBFOX, MBNL, CELF, hnRNP, or ESRP classes of splicing factors. The EMT alternative splicing signature was confirmed in human breast cancer cell lines, which could be classified into basal and luminal subtypes based exclusively on their EMT–associated splicing pattern. Expression of EMT–associated alternative mRNA transcripts was also observed in primary breast cancer samples, indicating that EMT–dependent splicing changes occur commonly in human tumors. The functional significance of EMT–associated alternative splicing was tested by expression of the epithelial-specific splicing factor ESRP1 or by depletion of RBFOX2 in mesenchymal cells, both of which elicited significant changes in cell morphology and motility towards an epithelial phenotype, suggesting that splicing regulation alone can drive critical aspects of EMT–associated phenotypic changes. The molecular description obtained here may aid in the development of new diagnostic and prognostic markers for analysis of breast cancer progression.

Subtle discrepancies of SF2/ASF ESE sequence motif among human tissues: A computational approach

The intron removal during the pre-mRNA splicing in higher eukaryotes requires the accurate identification of the two splice sites at the ends of the exons, or exon definition. However, the consensus sequences at the splice sites provide insufficient information to distinguish true splice sites from the large number of the false ones that populate the primary transcripts. Additional information is provided by cis-acting regulatory sequences that serve to enhance or repress splicing, and that may be exonic or intronic in nature: the splicing enhancers and the splicing silencers, respectively. In this study, we tested by computational and statistical approaches if the exonic splicing enhancer motif binding to the SF2/ASF SR protein is conserved among several groups of human genes. The results showed that the SF2/ASF ESE consensus was conserved between genes within the same chromosome, within different chromosomes and between different levels of muscular cells differentiation. However, this motif displays subtle variations within the consensus sequence between genes expressed in different tissues. These results can emphasize the presence of different translational isoforms of the SFRS1 gene encoding for the SF2/ASF, or different post-translational protein maturations in different tissues. This tissular discrepancy can also account for the alternative splicing of several genes between tissues.

G.P.14.12 Myotonic dystrophy unlinked to DM1 and DM2 mutations in three siblings

Myotonic dystrophy (DM) is the most common adult form of muscular dystrophy, and is characterized by a peculiar multisystemic involvement. DM is caused by pathogenic expansions of unstable CTG repeats in the DMPK gene (DM1), or CCTG repeats in the ZNF9 gene (DM2). According to current understanding, DM pathogenesis is related to intranuclear toxicity of the mutant RNA, resulting in the impairment of alternative splicing of several genes. Only a few DM cases lacking segregation with DM1 and DM2 loci have been reported so far.

Differential Expression of the B′β Regulatory Subunit of Protein Phosphatase 2A Modulates Tyrosine Hydroxylase Phosphorylation and Catecholamine Synthesis

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, is stimulated by N-terminal phosphorylation by several kinases and inhibited by protein serine/threonine phosphatase 2A (PP2A). PP2A is a family of heterotrimeric holoenzymes containing one of more than a dozen different regulatory subunits. In comparison with rat forebrain extracts, adrenal gland extracts exhibited TH hyperphosphorylation at Ser(19), Ser(31), and Ser(40), as well as reduced phosphatase activity selectively toward phosphorylated TH. Because the B'beta regulatory subunit of PP2A is expressed in brain but not in adrenal glands, we tested the hypothesis that PP2A/B'beta is a specific TH phosphatase. In catecholamine-secreting PC12 cells, inducible expression of B'beta decreased both N-terminal Ser phosphorylation and in situ TH activity, whereas inducible silencing of endogenous B'beta had the opposite effect. Furthermore, PP2A/B'beta directly dephosphorylated TH in vitro. As to specificity, other PP2A regulatory subunits had negligible effects on TH activity and phosphorylation in situ and in vitro. Whereas B'beta was highly expressed in dopaminergic cell bodies in the substantia nigra, the PP2A regulatory subunit was excluded from TH-positive terminal fields in the striatum and failed to colocalize with presynaptic markers in general. Consistent with a model in which B'beta enrichment in neuronal cell bodies helps confine catecholamine synthesis to axon terminals, TH phosphorylation was higher in processes than in somata of dopaminergic neurons. In summary, we show that B'beta recruits PP2A to modulate TH activity in a tissue- and cell compartment specific fashion.

Regulation of Ich-1 pre-mRNA alternative splicing and apoptosis by mammalian splicing factors.

The importance of alternative splicing in regulating apoptosis has been suggested by findings of functionally antagonistic proteins generated by alternative splicing of several genes involved in apoptosis. Among these, Ich-1 (also named as caspase-2) encodes a member of the caspase family of proteases. Two forms of Ich-1 are produced as a result of alternative splicing: Ich-1L, which causes apoptosis, and Ich-1S, which prevents apoptosis. The precise nature of Ich-1 alternative splicing and its regulation remain unknown. Here, we show that the production of Ich-1L and Ich-1S transcripts results from alternative exclusion or inclusion of a 61-bp exon. Several splicing factors can regulate Ich-1 splicing. Serine-arginine-rich proteins SC35 and ASF/SF2 promote exon skipping, decreasing the ratio of Ich-1S to Ich-1L transcripts; whereas heterogeneous nuclear ribonucleoprotein A1 facilitates exon inclusion, increasing this ratio. Furthermore, in cultured cells, SC35 overexpression increases apoptosis; whereas heterogeneous nuclear ribonucleoprotein A1 overexpression decreases apoptosis. These results provide the first direct evidence that splicing factors can regulate Ich-1 alternative splicing and suggest that alternative splicing may be an important regulatory mechanism for apoptosis.